Particle classification and sorting system and method

Abstract

In some examples, a method for processing particles in a particle stream is provided, the method including delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream comprising a particle stream including a plurality of particles, directing interrogation electromagnetic radiation at particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles, and then directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream to sort the particles into at least two populations depending on the monitored responsive optical emissions of the particles, the microfluidic stream comprising a continuous phase flow of a liquid.

Description

[Technical Field]

[0001] The present disclosure relates to sorting and separation of particles, such as asymmetric biological cells. [Background technology]

[0002] Sorting particles with different properties is useful for many downstream processes. For example, sorting sperm cells into populations X and Y allows for downstream separation or sorting of these two populations. For certain animal breeding, certain categories of sperm cells may be more desirable. For example, bovine X sperm cells are preferred for insemination of cattle to primarily produce female offspring for the milking herd.

[0003] Sorting and sorting bovine sperm cells presents challenges, including low sorting efficiency and slow sorting speed. Low sorting efficiency results in a low percentage of desired cells (e.g., X sperm cells) in the collection container compared to the total number of cells introduced into the sorting and sorting system. Low sorting efficiency can be caused by several factors, including poor orientation of cells for sorting, inaccurate sorting techniques, low-efficiency sorting techniques, and related processes that negatively affect cell motility. Slow sorting speeds extend the time that biological cells are outside of optimal storage conditions, and therefore may also affect cell motility.

[0004] Any reference herein to patent specifications, other external documents, or other sources of information is generally for the purpose of providing a context for discussing the inventive features disclosed herein. Unless otherwise expressly stated, the reference to such external documents should not be construed as an admission that such documents, or such sources of information, are prior art or form part of the common general knowledge in the art in any jurisdiction.

[0005] It is an object of the present invention to provide improved methods for sorting and / or separating particles having distinguishable characteristics, or at least to provide the public with a useful choice of particle sorting and / or separation methods. Summary of the Invention

[0006] In some examples, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic vapor from a microfluidic aperture to a flow environment, the microfluidic vapor comprising a particle stream including a plurality of particles, directing interrogation electromagnetic radiation at particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles, and then directing sorting electromagnetic radiation at at least some of the particles in the microfluidic stream to sort the particles into at least two populations depending on the monitored responsive optical emissions of the particles, wherein the microfluidic stream comprises a continuous phase flow of a liquid.

[0007] In one example, the particle stream may be surrounded by a sheath stream.

[0008] In one example, the flow environment includes one or more of a microchannel, optionally including a substantially transparent material, a substrate exposed to the fluid environment, a liquid fluid environment, a gaseous fluid environment, and the flow environment may include a gaseous fluid environment, substantially parallel to the microfluidic flow and at least partially surrounded by a gas sheath moving relative to the gaseous environment.

[0009] In one example, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to apply radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and directing subsequent sorting electromagnetic radiation at particles that are biological cells, the subsequent sorting electromagnetic radiation configured to deliver energy to the selected cells that is below a predetermined ablation threshold corresponding to rupturing a cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after the freezing and thawing process.

[0010] In one example, the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective lens.

[0011] In one example, the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective at an angle relative to one another.

[0012] In one example, the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through a common optical objective lens is adjusted to define an inter-beam distance between the focal point of the inspection electromagnetic radiation within the microfluidic vapor and the focal point of the selection electromagnetic radiation within the microfluidic vapor.

[0013] In one example, the microfluidic stream is delivered from a flow control device having a microfluidic aperture, the flow control device being shaped to define a region above the microfluidic aperture through which the inspection and / or screening electromagnetic radiation is directed, and an objective optical component may be positioned at least partially within the region.

[0014] In one example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a diverging distance from the microfluidic aperture, the diverging distance being between 25 and 1000 μm.

[0015] In one example, the divergence distance is less than 400 μm or greater than one of 25 μm, 50 μm, or 100 μm.

[0016] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0017] In one example, the interrogation electromagnetic radiation and / or the screening electromagnetic radiation are controlled to propagate as respective beams that are equal to or wider than the microfluidic stream when they intersect with the microfluidic stream.

[0018] In one example, monitoring the responsive luminescence from the illuminated particles includes using outputs from multiple sensors arranged around the microfluidic stream arranged to capture the responsive luminescence from different directions.

[0019] In one example, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0020] In one example, the method includes adjusting a cross-section of a microfluidic aperture.

[0021] In one example, the flow velocity of the microfluidic flow is 5 to 20 m / s.

[0022] In some examples, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream containing a plurality of particles, directing interrogation electromagnetic radiation through a common optical objective to particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles, and then directing sorting electromagnetic radiation through the common optical objective to at least some of the particles in the microfluidic stream to sort the particles into at least two populations depending on the monitored responsive optical emissions of the particles.

[0023] In one example, the particle stream may be surrounded by a sheath stream.

[0024] In some examples, the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective at an angle relative to one another.

[0025] In some examples, the angle between the inspection electromagnetic radiation and the screening electromagnetic radiation directed through the common optical objective lens is adjusted to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic stream and a focal point of the screening electromagnetic radiation within the microfluidic stream.

[0026] In some examples, the microfluidic stream is delivered from a flow control device having a microfluidic aperture, and the flow control device begins to be shaped to define a region above the microfluidic aperture through which the interrogation electromagnetic radiation and / or screening electromagnetic radiation is directed.

[0027] In some examples, the objective optical component is positioned at least partially within the region.

[0028] In some examples, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a diverging distance from the microfluidic aperture, the diverging distance being between 25 and 1000 μm.

[0029] In some examples, the divergence distance is less than 400 μm or greater than one of 25 μm, 50 μm, or 100 μm.

[0030] In some examples, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance that includes at least 10 μm, one or more of 10 μm to 400 μm.

[0031] In some examples, the interrogation electromagnetic radiation and / or the screening electromagnetic radiation is controlled to propagate as a screening beam that is equal to or wider than the microfluidic stream when it intersects with the microfluidic stream.

[0032] In some examples, monitoring the responsive emissions from the illuminated particles includes using outputs from multiple sensors arranged around the microfluidic stream using a photoarray with detectors positioned to capture the responsive emissions from different directions.

[0033] In some examples, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0034] In some examples, the method includes adjusting a cross-section of the microfluidic aperture.

[0035] In some examples, the flow velocity of the microfluidic flow is 5-20 m / s.

[0036] In some examples, the microfluidic flow comprises a continuous phase flow of a liquid.

[0037] In some examples, the flow environment includes one or more of the following: a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, and a gaseous fluid environment.

[0038] In some examples, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0039] In some examples, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to apply radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and directing subsequent sorting electromagnetic radiation at particles that are biological cells, the subsequent sorting electromagnetic radiation configured to deliver energy to the selected cells that is below a predetermined ablation threshold corresponding to rupturing a cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after the freezing and thawing process.

[0040] In some examples, a method for processing particles in a particle stream is provided, the method including delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream containing a plurality of particles, directing an inspection beam at particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles, and then sorting the particles into at least two populations dependent on the monitored responsive optical emissions of the particles, wherein a divergence distance between the aperture and the inspection beam is less than 1000 μm.

[0041] In one example, the particle stream may be surrounded by a sheath stream.

[0042] In some instances, the divergence distance is less than 400 μm.

[0043] In some examples, the divergence distance is greater than one of 25 μm, 50 μm, or 100 μm.

[0044] In some examples, the flow velocity of the microfluidic flow is 5-20 m / s.

[0045] In some examples, sorting the particles includes directing sorting electromagnetic radiation to at least a portion of the particles in the microfluidic stream to sort the particles into at least two populations.

[0046] In some examples, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to apply radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and directing subsequent sorting electromagnetic radiation at particles that are biological cells, the subsequent sorting electromagnetic radiation configured to deliver energy to the selected cells that is below a predetermined ablation threshold corresponding to rupturing a cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after the freezing and thawing process.

[0047] In some examples, the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective.

[0048] In some examples, the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective at an angle relative to one another.

[0049] In some examples, the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjusted to define an inter-beam distance between the focal point of the inspection electromagnetic radiation within the microfluidic vapor and the focal point of the selection electromagnetic radiation within the microfluidic vapor.

[0050] In some examples, the microfluidic stream is delivered from a flow control device having a microfluidic aperture, and the flow control device begins to be shaped to define a region above the microfluidic aperture through which the interrogation electromagnetic radiation and / or screening electromagnetic radiation is directed.

[0051] In some examples, the objective optical component is positioned at least partially within the region.

[0052] In some examples, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance that includes at least 10 μm, one or more of 10 μm to 400 μm.

[0053] In some examples, the interrogation electromagnetic radiation and / or the screening electromagnetic radiation is controlled to propagate as a screening beam that is equal to or wider than the microfluidic stream when it intersects with the microfluidic stream.

[0054] In some examples, the microfluidic flow comprises a continuous phase flow of a liquid.

[0055] In some examples, the flow environment includes one or more of the following: a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, and a gaseous fluid environment.

[0056] In some examples, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0057] In some examples, monitoring the responsive emission from the illuminated particles includes using outputs from multiple sensors arranged around the microfluidic stream using a photoarray having detectors positioned to capture the responsive emission from different directions.

[0058] In some examples, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0059] In some examples, the method includes adjusting a cross-section of the microfluidic aperture.

[0060] In some instances, a corresponding apparatus is provided.

[0061] In one example, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream and a surrounding sheath flow, the particle stream including a plurality of particles; directing interrogation electromagnetic radiation at particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles; and sorting the particles into at least two populations depending on the monitored responsive optical emissions of the particles, wherein the microfluidic aperture has a cross-section that extends more in one axis than a perpendicular axis.

[0062] In one example, the microfluidic flow includes one or more of the following: a continuous phase flow of liquid, and a dispersed flow of droplets.

[0063] In one example, the flow environment includes one or more of a microchannel, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

[0064] In one example, the flow environment includes a gaseous fluid environment, substantially parallel to the microfluidic flow, and is at least partially surrounded by a gas sheath that moves relative to the gaseous environment.

[0065] In one example, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0066] In one example, the interrogation electromagnetic radiation and the subsequent screening electromagnetic radiation propagate through a common objective lens.

[0067] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0068] In one example, the screening electromagnetic radiation is controlled to propagate as a screening beam that intersects with the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0069] In one example, the sorting beam is wider than the microfluidic stream.

[0070] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0071] In one example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

[0072] In one example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0073] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0074] In one example, the particles are biological cells, and the subsequent selective electromagnetic radiation is configured to deliver energy to the selected cells below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after the freezing and thawing process.

[0075] In one example, the microfluidic aperture has a cross section having one of the following shapes: ellipse, rectangle, parallelogram, trapezoid, polygon, square.

[0076] In one example, the ratio of the cross-sectional extension of the microfluidic aperture in one axis to the cross-sectional extension of the microfluidic aperture in a perpendicular axis is 1:100 and 2:3.

[0077] In one example, the ratio is 1:50 to 1:10.

[0078] In one example, the microfluidic aperture has an adjustable cross-section.

[0079] In one example, the cross-sectional size and / or shape of the microfluidic aperture is adjustable.

[0080] In one example, the cross section is automatically adjusted according to a performance metric associated with the microfluidic flow.

[0081] In one example, the wash mode is characterized by a maximum cross-sectional area of ​​the microfluidic aperture and a wash flow of liquid through the microfluidic aperture, optionally with the wash flow having a higher flow rate than the microfluidic flow.

[0082] In one example, the method includes directing concentrated electromagnetic radiation at the sheath flow to vaporize a portion of the microfluidic flow, subsequent to directing the interrogation electromagnetic radiation.

[0083] In one example, the concentrated electromagnetic radiation is controlled depending on the monitored responsive emission of the particles.

[0084] In one example, monitoring the responsive emission of light from the illuminated particles includes using outputs from a plurality of sensors arranged around the microfluidic stream.

[0085] In one example, the multiple sensors are implemented as a photoarray with detectors positioned to capture responsive emissions from different directions.

[0086] In one example, the output from the sensor is adjusted depending on the position of the sensor.

[0087] In one example, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0088] In one example, the plurality of sensors is arranged in a plane perpendicular to the longitudinal axis of the microfluidic stream, and the plurality of sensors is arranged in an arc around the microfluidic stream or in a substantially straight line adjacent to the microfluidic stream.

[0089] In one example, outputs from sensors corresponding to responsive emissions from particles are integrated to generate signals that are used to classify the particles.

[0090] In one example, the sheath extends parallel to the microfluidic stream and at least partially surrounds the microfluidic stream and the fluid environment.

[0091] In one example, the sheath includes one or more of the following: a gas flow that moves relative to the fluid environment and through which the interrogation electromagnetic radiation is directed; and a transparent solid material through which the interrogation electromagnetic radiation is directed.

[0092] In one example, the microfluidic aperture is defined in a flow controller that is used to generate the microfluidic flow.

[0093] In one example, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream including a particle stream and a surrounding sheath stream, the particle stream including a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic vapor and monitoring responsive optical emissions from the irradiated particles; sorting the particles into at least two populations dependent on the monitored responsive optical emissions of the particles; and, subsequent to directing the interrogation electromagnetic radiation, directing focused electromagnetic radiation at the sheath stream to vaporize a portion of the microfluidic stream.

[0094] In one example, the microfluidic flow includes one or more of the following: a continuous phase flow of liquid, and a dispersed flow of droplets.

[0095] In one example, the flow environment includes one or more of a microchannel, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

[0096] In one example, the flow environment includes a gaseous fluid environment, substantially parallel to the microfluidic flow, and is at least partially surrounded by a gas sheath that moves relative to the gaseous environment.

[0097] In one example, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0098] In one example, the interrogation electromagnetic radiation and the subsequent screening electromagnetic radiation propagate through a common objective lens.

[0099] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0100] In one example, the screening electromagnetic radiation is controlled to propagate as a screening beam that intersects with the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0101] In one example, the sorting beam is wider than the microfluidic stream.

[0102] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0103] In one example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

[0104] In one example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0105] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0106] In one example, the particles are biological cells, and the subsequent screening electromagnetic radiation is configured to deliver energy to the selected cells below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cells becoming non-viable after the freezing and thawing process.

[0107] In one example, the microfluidic aperture has a cross section that extends in one axis more than in a perpendicular axis.

[0108] In one example, the microfluidic aperture has a cross section having one of the following shapes: ellipse, rectangle, parallelogram, trapezoid, polygon.

[0109] In one example, the ratio of the cross-sectional extension of the microfluidic aperture in one axis to the cross-sectional extension of the microfluidic aperture in a perpendicular axis is 1:100 and 2:3.

[0110] In one example, the ratio is 1:50 to 1:10.

[0111] In one example, the microfluidic aperture has an adjustable cross-section.

[0112] In one example, the cross-sectional size and / or shape of the microfluidic aperture is adjustable.

[0113] In one example, the cross section is automatically adjusted according to a metric associated with the microfluidic flow.

[0114] In one example, the wash mode is characterized by a maximum cross-sectional area of ​​the microfluidic aperture and a wash flow of liquid through the microfluidic aperture, optionally with the wash flow having a higher flow rate than the microfluidic flow.

[0115] In one example, the concentrated electromagnetic radiation is controlled depending on the monitored responsive emission of the particles.

[0116] In one example, monitoring the responsive emission of light from the illuminated particles includes using outputs from a plurality of sensors arranged around the microfluidic stream.

[0117] In one example, the multiple sensors are implemented as a photoarray with detectors positioned to capture responsive emissions from different directions.

[0118] In one example, the output from the sensor is adjusted depending on the position of the sensor.

[0119] In one example, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0120] In one example, the plurality of sensors is arranged in a plane perpendicular to the longitudinal axis of the microfluidic stream, and the plurality of sensors is arranged in an arc around the microfluidic stream or in a substantially straight line adjacent to the microfluidic stream.

[0121] In one example, outputs from sensors corresponding to responsive emissions from particles are integrated to generate signals that are used to classify the particles.

[0122] In one example, the sheath extends parallel to the microfluidic stream and at least partially surrounds the microfluidic stream and the fluid environment.

[0123] In one example, the sheath includes one or more of the following: a gas flow that moves relative to the fluid environment and through which the interrogation electromagnetic radiation is directed; and a transparent solid material through which the interrogation electromagnetic radiation is directed.

[0124] In one example, the microfluidic aperture is defined in a flow controller that is used to generate the microfluidic flow.

[0125] In one example, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream and a surrounding sheath flow, the particle stream including a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles; and sorting the particles into at least two populations depending on the monitored responsive optical emissions of the particles, wherein monitoring the responsive optical emissions from the illuminated particles includes using outputs from a plurality of sensors arranged about the microfluidic stream.

[0126] In one example, the microfluidic flow includes one or more of the following: a continuous phase flow of liquid, and a dispersed flow of droplets.

[0127] In one example, the flow environment includes one or more of a microchannel, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

[0128] In one example, the flow environment includes a gaseous fluid environment, substantially parallel to the microfluidic flow, and is at least partially surrounded by a gas sheath that moves relative to the gaseous environment.

[0129] In one example, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles, directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles, and applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0130] In one example, the interrogation electromagnetic radiation and the subsequent screening electromagnetic radiation propagate through a common objective lens.

[0131] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0132] In one example, the screening electromagnetic radiation is controlled to propagate as a screening beam that intersects with the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0133] In one example, the sorting beam is wider than the microfluidic stream.

[0134] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0135] In one example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

[0136] In one example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0137] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0138] In one example, the particles are biological cells, and the subsequent screening electromagnetic radiation is configured to deliver energy to the selected cells below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cells becoming non-viable after the freezing and thawing process.

[0139] In one example, the microfluidic aperture has a cross section that extends in one axis more than in a perpendicular axis.

[0140] In one example, the microfluidic aperture has a cross section having one of the following shapes: ellipse, rectangle, parallelogram, trapezoid, polygon.

[0141] In one example, the ratio of the cross-sectional extension of the microfluidic aperture in one axis to the cross-sectional extension of the microfluidic aperture in a perpendicular axis is between 1:100 and 2:3.

[0142] In one example, the ratio is 1:50 to 1:10.

[0143] In one example, the microfluidic aperture has an adjustable cross-section.

[0144] In one example, the cross-sectional size and / or shape of the microfluidic aperture is adjustable.

[0145] In one example, the cross section is automatically adjusted according to a metric associated with the microfluidic flow.

[0146] In one example, the method includes a wash mode characterized by a maximum cross-sectional area of ​​the microfluidic aperture and a wash flow of liquid through the microfluidic aperture, optionally the wash flow having a higher flow rate than the microfluidic flow.

[0147] In one example, the concentrated electromagnetic radiation is controlled depending on the monitored responsive emission of the particles.

[0148] In one example, the method includes directing concentrated electromagnetic radiation at the sheath flow to vaporize a portion of the microfluidic flow, subsequent to directing the interrogation electromagnetic radiation.

[0149] In one example, the multiple sensors are implemented as a photoarray with detectors positioned to capture responsive emissions from different directions.

[0150] In one example, the output from the adjustment is scaled depending on the position of the sensor.

[0151] In one example, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0152] In one example, the plurality of sensors is arranged in a plane perpendicular to the longitudinal axis of the microfluidic stream, and the plurality of sensors is arranged in an arc around the microfluidic stream or in a substantially straight line adjacent to the microfluidic stream.

[0153] In one example, outputs from sensors corresponding to responsive emissions from particles are integrated to generate signals that are used to classify the particles.

[0154] In one example, the sheath extends parallel to the microfluidic stream and at least partially surrounds the microfluidic stream and the fluid environment.

[0155] In one example, the sheath includes one or more of the following: a gas flow that moves relative to the fluid environment and through which the interrogation electromagnetic radiation is directed; and a transparent solid material through which the interrogation electromagnetic radiation is directed.

[0156] In one example, the microfluidic aperture is defined in a flow controller that is used to generate the microfluidic flow.

[0157] In one example, a method for processing particles in a particle stream is provided, the method including: delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream and a surrounding sheath flow, the particle stream including a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles; sorting the particles into at least two populations dependent on the monitored responsive optical emissions of the particles; and generating a sheath including a gas flow moving relative to the fluid environment, extending substantially parallel to the microfluidic stream, and at least partially surrounding the microfluidic stream and the fluid environment.

[0158] In one example, the microfluidic flow includes one or more of the following: a continuous phase flow of liquid, and a dispersed flow of droplets.

[0159] In one example, the flow environment includes one or more of a microchannel, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

[0160] In one example, the flow environment includes a gaseous fluid environment, substantially parallel to the microfluidic flow, and is at least partially surrounded by a gas sheath that moves relative to the gaseous environment.

[0161] In one example, the sorting includes one or more of the following: directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic stream to ablate the particles; directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; and applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0162] In one example, the interrogation electromagnetic radiation and the subsequent screening electromagnetic radiation propagate through a common objective lens.

[0163] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0164] In one example, the screening electromagnetic radiation is controlled to propagate as a screening beam that intersects with the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0165] In one example, the sorting beam is wider than the microfluidic stream.

[0166] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0167] In one example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

[0168] In one example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0169] In one example, the inspection electromagnetic radiation is controlled to propagate as an ultraviolet or infrared inspection beam.

[0170] In one example, the particles are biological cells, and the subsequent selective electromagnetic radiation is configured to deliver energy to the selected cells below a predetermined ablation threshold corresponding to rupture of the cell membrane above a predetermined priming threshold corresponding to the cells becoming immobile after the freezing and thawing process.

[0171] In one example, the microfluidic aperture has a cross section that extends in one axis more than in a perpendicular axis.

[0172] In one example, the microfluidic aperture has a cross section having one of the following shapes: ellipse, rectangle, parallelogram, trapezoid, polygon.

[0173] In one example, the ratio of the cross-sectional extension of the microfluidic aperture in one axis to the cross-sectional extension of the microfluidic aperture in a perpendicular axis is between 1:100 and 2:3.

[0174] In one example, the ratio is 1:50 to 1:10.

[0175] In one example, the microfluidic aperture has an adjustable cross-section.

[0176] In one example, the cross-sectional size and / or shape of the microfluidic aperture is adjustable.

[0177] In one example, the cross section is automatically adjusted according to a metric associated with the microfluidic flow.

[0178] In one example, the wash mode is characterized by a maximum cross-sectional area of ​​the microfluidic aperture and a wash flow of liquid through the microfluidic aperture, the wash flow having a higher flow rate than the microfluidic flow.

[0179] In one example, the concentrated electromagnetic radiation is controlled depending on the monitored responsive emission of the particles.

[0180] In one example, monitoring the responsive emission of light from the illuminated particles includes using outputs from a plurality of sensors arranged around the microfluidic stream.

[0181] In one example, the multiple sensors are implemented as a photoarray with detectors positioned to capture responsive emissions from different directions.

[0182] In one example, the output from the sensor is adjusted depending on the position of the sensor.

[0183] In one example, the output from the sensors is normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0184] In one example, the sensors are arranged in a plane perpendicular to the longitudinal axis of the microfluidic stream, and the sensors are arranged in an arc around the microfluidic stream or in a substantially straight line adjacent to the microfluidic stream.

[0185] In one example, outputs from sensors corresponding to responsive emissions from particles are integrated to generate signals that are used to classify the particles.

[0186] In one example, the method includes directing concentrated electromagnetic radiation at the sheath flow to vaporize a portion of the microfluidic flow, subsequent to directing the interrogation electromagnetic radiation.

[0187] In one example, the interrogating electromagnetic radiation is directed through a sheath.

[0188] In one example, the microfluidic aperture is defined in a flow controller that is used to generate the microfluidic flow.

[0189] In one example, an apparatus for processing particles in a particle stream is provided, the apparatus including: means for delivering a microfluidic stream from a microfluidic aperture to a flow environment, the microfluidic stream including a particle stream and a surrounding sheath stream, the particle stream including a plurality of particles; means for directing interrogation electromagnetic radiation at the particles in the microfluidic vapor and monitoring responsive light emissions from the illuminated particles; and means for sorting the particles into at least two populations dependent on the monitored responsive light emissions of the particles, wherein the microfluidic aperture has a cross-section extending along an axis other than a normal axis; and / or the means for monitoring the responsive light emissions from the illuminated particles uses output from sensors arranged about the microfluidic stream; and / or the apparatus includes means for directing concentrated electromagnetic radiation at the sheath stream to vaporize a portion of the microfluidic stream following the directing of the interrogation electromagnetic radiation; and / or the apparatus includes means for generating a sheath moving relative to the fluid environment, extending substantially parallel to the microfluidic stream and including a gas flow at least partially surrounding the microfluidic stream and the fluid environment.

[0190] In one example, the microfluidic flow includes one or more of the following: a continuous phase flow of liquid, and a dispersed flow of droplets.

[0191] In one example, the flow environment includes one or more of a microchannel, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

[0192] In one example, the flow environment includes a gaseous fluid environment, substantially parallel to the microfluidic flow, and is at least partially surrounded by a gas sheath that moves relative to the gaseous environment.

[0193] In one example, the means for sorting includes one or more of the following: means for directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart radiation pressure to the particles; means for directing subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; means for applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0194] In one example, the inspection electromagnetic radiation and the subsequent selection electromagnetic radiation are arranged to propagate through a common objective lens.

[0195] In one example, the inspection electromagnetic radiation and subsequent screening electromagnetic radiation are arranged to propagate as respective inspection beams and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of 10 μm to 400 μm.

[0196] In one example, the screening electromagnetic radiation is arranged to propagate as a screening beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0197] In one example, the sorting beam is wider than the microfluidic stream.

[0198] In one example, the inspection electromagnetic radiation is arranged to propagate as an ultraviolet or infrared inspection beam.

[0199] In one example, the interrogation electromagnetic radiation is arranged to propagate as an interrogation beam that intersects the microfluidic stream at a diverging distance from the microfluidic aperture, the diverging distance being between 25 and 1000 μm.

[0200] In one example, the interrogation electromagnetic radiation is arranged to propagate as an interrogation beam that intersects the microfluidic stream and provides an elliptical or circular intensity pattern within the microfluidic stream.

[0201] In one example, the inspection electromagnetic radiation is arranged to propagate as an ultraviolet or infrared inspection beam.

[0202] In one example, the particles are biological cells, and the subsequent selective electromagnetic radiation is configured to deliver energy to the selected cells below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after the freezing and thawing process.

[0203] In one example, the microfluidic aperture has a cross section having one of the following shapes: ellipse, rectangle, parallelogram, trapezoid, polygon.

[0204] In one example, the ratio of the cross-sectional extension of the microfluidic aperture in one axis to the cross-sectional extension of the microfluidic aperture in a perpendicular axis is between 1:100 and 2:3.

[0205] In one example, the ratio is 1:50 to 1:10.

[0206] In one example, the microfluidic aperture has an adjustable cross-section.

[0207] In one example, the cross-sectional size and / or shape of the microfluidic aperture is adjustable.

[0208] In one example, the device is configured to automatically adjust the cross section in response to a performance metric associated with the microfluidic flow.

[0209] In one example, the device is configured to operate in a wash mode characterized by a maximum cross-sectional area of ​​the microfluidic aperture and a wash flow of liquid through the microfluidic aperture, optionally the wash flow having a higher flow rate than the microfluidic flow.

[0210] In one example, the concentrated electromagnetic radiation is controlled depending on the monitored responsive emission of the particles.

[0211] In one example, the multiple sensors are implemented as a photoarray with detectors positioned to capture responsive emissions from different directions.

[0212] In one example, the output from the sensor is adjusted depending on the position of the sensor.

[0213] In one example, the outputs from the sensors are arranged to be normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refractive effects when traversing from the microfluidic flow to the flow environment.

[0214] In one example, the plurality of sensors is arranged in a plane perpendicular to the longitudinal axis of the microfluidic stream, and the plurality of sensors is arranged in an arc around the microfluidic stream or in a substantially straight line adjacent to the microfluidic stream.

[0215] In one example, outputs from sensors corresponding to responsive emissions from particles are integrated to generate signals that are used to classify the particles.

[0216] In one example, the device comprises a second sheath extending parallel to the microfluidic stream and at least partially enclosing the microfluidic stream and the fluid environment.

[0217] In one example, the second sheath includes one or more of the following: a gas flow that moves relative to the fluid environment and through which the interrogation electromagnetic radiation is directed; and a transparent solid material through which the interrogation electromagnetic radiation is directed.

[0218] In one example, the microfluidic aperture is defined in a flow controller that is used to generate the microfluidic flow.

[0219] Aspects of the invention may also be said to reside broadly in the parts, elements and features referred to or shown in the specification of the application, individually or collectively, in any or all combinations of two or more of the parts, elements or features, and where specific integers that have known equivalents in the art to which the invention pertains are referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth. [Brief explanation of the drawings]

[0220] The present invention will now be described, by way of example only, with reference to the following drawings.

[0221] [Figure 1] 1 is a schematic diagram of a system for sorting and separating particles, according to some examples. [Figure 2] 1 is a schematic diagram of a portion of a system for sorting and separating particles, according to some examples. [Figure 3] 1 illustrates a longitudinal cross section of a flow control device for use with a system for sorting and separating particles, according to some examples. [Figure 4] 4 shows a cross-sectional view of the flow control device of FIG. 3. [Figure 5] 1 illustrates an aperture of a delivery tube for use with a system for sorting and separating particles, according to some examples. [Figure 6] 1 illustrates the use of surface tension to improve particle orientation, according to an embodiment. [Figure 7] 1 illustrates an aperture of a delivery tube for use with a system for sorting and separating particles, according to some examples. [Figure 8] 1 illustrates an adjustable aperture in a delivery tube for use with a system for sorting and separating particles, according to some embodiments. [Figure 9] 1 illustrates a side view of an adjustable aperture of a delivery tube for use with a system for sorting and separating particles, according to some examples. [Figure 10a]1 illustrates the intensity distribution of a beam for inspecting or sorting particles, according to some examples. [Figure 10b] 1 illustrates the intensity distribution of a beam for inspecting or sorting particles, according to some examples. [Figure 10c] 1 illustrates the intensity distribution of a beam for inspecting or sorting particles, according to some examples. [Figure 11] 1 illustrates the intensity field of a beam focused at a nominal focal point X according to an example. [Figure 12] 1 illustrates an intensity region of a beam focused off-center of a microfluidic flow according to an example. [Figure 13] An example shows the beam distance vs. flow velocity. [Figure 14] 1 illustrates a sheath with separate sheath components for an objective lens according to an example. [Figure 15] 1 illustrates an implementation of a vaporization device according to an example. [Figure 16a] 1 illustrates droplet formation regulation with some examples. [Figure 16b] 1 illustrates droplet formation regulation with some examples. [Figure 17a] 1 illustrates a detection device according to some examples. [Figure 17b] 1 illustrates a detection device according to some examples. [Figure 18] 1 shows an example of the present invention showing a trumpet shaped aperture. [Figure 19] 1 shows an example showing a tapered microfluidic delivery tube. [Figure 20a] 1 illustrates an example of a flow control device shaped to define a region upstream from an aperture through which at least a portion of the inspection beam and / or selection beam is directed to pass and / or in which an objective lens is at least partially disposed. [Figure 20b] 1 shows an example illustrating the stabilization of a microfluidic stream emitted from an aperture. [Figure 21a] The effect of divergence distance on particle identification resolution is shown. [Figure 21b] The effect of divergence distance on particle identification resolution is shown. [Figure 22a] 1 shows an example showing an inspection beam and a selection beam propagating towards a common objective lens at an angle relative to each other. [Figure 22b] 1 shows an example showing an inspection beam and a selection beam propagating towards a common objective lens at an angle relative to each other. [Figure 22c] 1 shows an example showing an inspection beam and a selection beam propagating towards a common objective lens at an angle relative to each other. [Figure 23] 10 illustrates an example of using an arrangement of optical components to provide angled inspection and selection beams. [Figure 24a] 10 shows an example of a beam splitter arrangement for providing angled inspection and selection beams. [Figure 24b] 10 shows an example of a beam splitter arrangement for providing angled inspection and selection beams. [Figure 24c] 10 shows an example of a beam splitter arrangement for providing angled inspection and selection beams. DETAILED DESCRIPTION OF THE INVENTION

[0222] In the claims, as well as in the above specification, all transitional phrases such as "comprise," "include," "carry," "have," "contain," "accompany," "hold," "consisting of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0223] As used herein, the term "about" refers to a reasonable amount of deviation from the modified term such that the end result will not be significantly altered. For example, when applied to a value, the term should be interpreted as including a deviation of + / - 5% of that value.

[0224] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0225] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0226] The terms "can" and "may" are used interchangeably in this disclosure to indicate that a mentioned element, component, structure, feature, functionality, purpose, advantage, operation, step, process, apparatus, system, device, result, or description has the capability to be used, included, or produced, or otherwise express a proposition indicated in the description where the term is used (or referred to) for a specific example.

[0227] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer, in one example, to A only (optionally including elements other than B); in another example, to B only (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements), etc.

[0228] As used herein and in the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one example, to the inclusion of at least one, optionally one or more A, with no B (and optionally including elements other than B); in another example, to the inclusion of at least one, optionally one or more B, with no A (and optionally including elements other than A); and, in yet another example, to the inclusion of at least one, optionally one or more A, and at least one, optionally one or more B (and optionally including other elements).

[0229] Reference to a range of numbers disclosed herein (e.g., 1 to 10) also incorporates reference to every rational number within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), and also incorporates reference to any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7); thus, all subranges of every range explicitly disclosed herein are intended to be expressly disclosed hereby. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly set forth in this application in the same manner.

[0230] Whenever a range is given herein, e.g., a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are intended to be included in the disclosure.

[0231] The following describes specific details, such as particular examples, or examples for purposes of explanation rather than limitation. Those skilled in the art will understand that other examples may be employed apart from these specific details. In some examples, detailed descriptions of well-known methods, nodes, interfaces, circuits, and devices are omitted so as not to obscure the description with unnecessary detail. Those skilled in the art will understand that the described functionality may be implemented in one or more nodes using hardware circuits (e.g., analog and / or discrete logic gates interconnected to perform specialized functions, ASICs, PLAs, etc.) and / or using software programs and data in conjunction with one or more digital microprocessors or general-purpose computers. Nodes that communicate using an air interface also have appropriate wireless communication circuitry. Furthermore, where appropriate, the technology may further be considered to be embodied entirely within any form of computer-readable memory, such as a solid-state memory, a magnetic disk, or an optical disk, containing an appropriate set of computer instructions that cause a processor to execute the technology described herein.

[0232] A hardware implementation may include or encompass hardware (e.g., digital or analog) circuitry, including, but not limited to, digital signal processor (DSP) hardware, reduced instruction set processors, application specific integrated circuits (ASICs) and / or field programmable gate arrays (FPGAs), and (where appropriate) state machines capable of performing such functions. Memory may be used for storing temporary variables, holding and transferring data between processes, non-volatile configuration settings, standard messaging formats, etc. Any suitable form of volatile and non-volatile storage may be employed, including random access memory (RAM) implemented as a metal-oxide semiconductor (MOS) or integrated circuit (IC), as well as storage implemented as hard disk drives and flash memory.

[0233] Some or all of the described devices or functionality may be instantiated in a cloud environment such as Docker, Kubernetes, or Spark. This cloud functionality may be instantiated at the network edge, device edge, local premises, or on a remote server coupled over a network such as 4G or 5G. Alternatively, this functionality may be implemented in dedicated hardware.

[0234] The term "confinement" as referred to herein refers to the restriction of the cross-sectional shape and size of a particle stream in a fluid stream. For example, the diameter of a circular cross-section of a stream may be restricted, or the major and minor dimensions of a stream of elliptical cross-section may be restricted, which may result in a single, narrow trajectory with minimal deviation in any polar axis of the particle from the defined central longitudinal axis of the stream.

[0235] The term "orientation" of asymmetric particles (including cells) refers to the predominant angle of the faces of a representative sample of particles relative to an axis substantially perpendicular to the axis of particle flow. In the absence of features that impart an orienting torque to the particles, the face orientations are expected to be randomly distributed and point at any angle within approximately 360°. A sample of cells to which an orienting torque is applied via an orienting feature will have a non-random angular orientation that preferentially orients the faces of the particles at a particular angle, such that the predominant angle can be determined or observed.

[0236] "Cells" and "X cells" are referred to herein as examples of particular types of particles that may be desirable to retain within a microfluidic sorting device. Where the term cell is used herein, it may be substituted with the term "particle," and cells / particles need not be living cells. Those skilled in the art will readily understand that reference to X cells is intended to refer to any other cell or particle having characteristics suitable for examination and sorting in accordance with the present invention. In particular, X cells may be used herein in place of any type of particle or cell, including substantially symmetric and asymmetric cells, neurons, red blood cells, tagged cells, viruses, or microbiota, as known to those skilled in the art.

[0237] The term "microfluidic flow" as referred to herein refers to a liquid flow having at least one geometrically constrained dimension where surface forces dominate volumetric forces. In one example, this can include a liquid flow having a submillimeter diameter or other cross-sectional dimension. In one example, a microfluidic flow can be a continuous-phase flow of a liquid, such as an uninterrupted flow of one or more aqueous solutions. It can be a laminar flow having a particle flow containing particles and a sheath flow surrounding the particle flow. Alternatively or additionally, a microfluidic flow can include a dispersed flow of droplets. A microfluidic flow can be associated with one or more performance metrics, such as flow rate, cross-sectional diameter and / or size, distance to droplet formation, etc.

[0238] The term "flow environment" as referred to herein refers to an environment in which a microfluidic stream can flow. One example is a microchannel, which may include a material such as glass that forms an elongated lumen or pathway through which the microfluidic stream flows. The pathway may be completely contained by the material between each end of the pathway, or the pathway may have at least one boundary that exposes the microfluidic stream to the fluid environment, with the material forming a substrate that interfaces with other boundaries of the microfluidic stream. In another example, the flow environment may be substantially stationary or may itself be a fluid environment or volume that can flow. In this example, the microfluidic stream does not interface with a material substrate but may be completely contained by the fluid environment. The fluid environment may be a liquid, such as an aqueous solution, or a gas, such as air.

[0239] FIG. 1 illustrates a sorting system 100 that includes a preparation station 105 that delivers prepared particles to a flow controller 110, which delivers the particles to a microfluidic stream 115 for downstream processing. The microfluidic stream 115 may be a laminar flow having a range of cross-sectional dimensions and carried within a flow environment. In one example, the flow environment may include a volume of gas, such as air, or a microchannel that completely or partially surrounds the microfluidic channel. One or more illuminators 120, such as infrared (IR) or ultraviolet (UV) illuminators or other illumination devices, generate an inspection beam. The inspection beam illuminates particles within the microfluidic stream in an inspection region 125. Illumination of the particles causes the particles to emit an illumination pattern, such as scattered light or fluorescence, that is detected by one or more detectors 130. Measured characteristics of the detected illumination pattern generate one or more signals that are forwarded to an analysis unit 135.

[0240] The analysis unit 135 may comprise a processor and memory and is configured to interpret these signals and control a sorting device 140 that processes particles in the different populations P1 and P2 depending on an analysis of the signals associated with those particles. An exemplary analysis unit 135 is described in International Patent Publication No. WO 2022139597(A1), which is incorporated herein by reference.

[0241] If the analysis unit 135 determines that a particle has a particular classification based on the analysis of its respective measurement signal, the sorting device 140 is controlled to sort this particle. Various sorting methods can be used to select this subpopulation of particles.

[0242] The illuminator 120 includes an excitation source adapted to direct interrogation electromagnetic radiation to generate an "interrogation beam" and induce signal or pattern emission from particles, such as cells, e.g., stained cells. In one example, the illuminator comprises an infrared (IR) or mid-infrared laser 123, more preferably a mid-infrared quantum cascade laser (QCL). These lasers have the potential to focus sufficient energy onto a single cell to perform accurate and rapid measurements. QCLs offer several advantages over conventional mid-infrared sources, including delivering very high spectral power densities and very high spatial or angular power densities. This allows QCLs to deliver 10,000,000 times more effective mid-infrared power to a single cell than conventional mid-infrared sources. QCLs also enable detection of cells with lower levels of staining, or in some examples, label-free detection. Dyes or labels may alter or damage cells. Therefore, when using label-free detection of cells, measurements using mid-infrared illumination are 25 times lower in energy than those used in FACS, eliminating photon damage and enabling high-throughput (>10,000 cells / sec) capabilities. Mid-infrared can include wavelengths from 5 to 28 microns.

[0243] In an alternative example, the illuminator 120 generates a UV laser 123, which may be pulsed, continuous wave, or quasi-continuous wave. The illuminator may be an LED, such as a UV LED. In one example, the illuminator generates an inspection beam 123 having a wavelength of 300-400 nm, and in a particular example, a wavelength of 355 nm. In one alternative or additional mode, the beam frequency may be continuous wave to 100 MHz. In another alternative or additional aspect, the beam power is between 1 mW and 1 Watt.

[0244] To achieve beam focusing and / or spatial shaping, focusing optics are included in the system.

[0245] The sorting device 140 may include a radiation source (or nudging laser) configured to direct radiation at particles to produce at least one of a force and a torque on each particle so as to induce at least one of a displacement and an orientation (or nudge) of each particle relative to an axis defined by the direction of the fluid flow. In examples, the system also includes at least one of free-space optics, optical fibers, and other waveguides configured to direct radiation from the radiation source onto the microfluidic fluid flow. In examples, the radiation source of the sorting device 140 may include a laser and be configured for strobe operation. In examples described herein, this emission or direction of sorting electromagnetic radiation (also referred to herein as "radiation pressure") from the radiation source is referred to as a "sorting beam." Some examples may utilize a mid-infrared beam, such as those described above, for use as an illuminator.

[0246] An implementation of the sorter may include a radiation source used to "nudge" particles based on the classification (e.g., P1 or P2) determined by the analyzer 135 using the output of the detector 130. In one example, the microfluidic stream is contained within a microchannel, and the nudged particulates then travel until they reach a branch 145. In some examples, the continuous fluid stream may be split into two or more branches corresponding to two or more separate collection tubes 160. With two collection branches, as shown in FIG. 1 , it can be appreciated that the stream 115 may be split into three, four, five, or more separate collection branches. The number of collection branches may be enabled by the use of a specific configuration of the sorter 140, which may include baffles, vanes, and / or branching elements 145 positioned within the microfluidic stream 115. This is further understood when considering a microchannel as a three-dimensional structure. Additionally, the sorter 140 may include two or more radiation sources, optionally positioned on either side of the microfluidic stream and separately controllable. These radiation sources "nudge" particles in multiple directions within the microfluidic flow. In one example, the arbitrarily positioned radiation sources may be multiple radiation sources arranged around the circumference of a circular cross-section microfluidic channel, where the circumferentially arranged radiation sources allow for precise control of particles in any direction. An exemplary sorting device is described in International Patent Publication No. 2020 / 013903(A2), which is incorporated herein by reference.

[0247] Those skilled in the art will appreciate that alternative methods can be used within a sorting device to achieve separation of particles with desired properties. For example, the use of electrostatic sorting or microbubble-induced particle sorting is known to those skilled in the art and is designed to accomplish the same task. Here, microbubbles or droplets containing nudged particles may move transversely to an axis corresponding to the direction of flow and not necessarily remain entrained within the separated fluid streams. Increasing the power of the radiation source, or similar alternative modifications of the sorting device, can increase the initial separation distance between microparticle streams, but the ability to do so without affecting cell viability, for example, may be desirable. Therefore, using this sorting method, careful control of the radiation source is desirable to ensure that selected cells are displaced into a different flow path compared to unselected cells and that the selected cells are not rendered immobile or nonviable as a result of the radiation. This displacement effect is preferably achieved by applying electromagnetic radiation, e.g., by a laser, to redirect the cells from a first flow path to a different flow path. The particle flow path containing the selected (P1) or unselected (P2) cells can then be directed to a first collection vessel, and the particle flow containing the other subpopulation of cells can be discarded or collected in a second, different collection vessel.

[0248] In another example, the sorting device 140 includes an electromagnetic radiation source that irradiates the microfluidic stream to achieve ablation or damage of selected particles therein. In this example, the emission or direction of the sorting electromagnetic radiation from the radiation source that causes ablation is also referred to as a "sorting beam." This arrangement is particularly useful for removing undesired cells within a larger population. For example, during the production of a cell population for CART cell therapy, certain types of cells may be present that do not exhibit a desired phenotype. Cells within this first population (P1) are destroyed, denatured, or rendered immobile by the sorting device. Alternate populations that do not exhibit the desired characteristics in the microfluidic stream 115, such as desirable cells not selected by the analysis unit, remain undisturbed (P2). The sorted or processed cells in the microfluidic stream 115 can then be collected in one or more collection vessels 160 for further use. The sorting device 140 thereby provides a cell population (P2) enriched for the desired characteristics. When using sperm cells, this desired population may include motile X cells.

[0249] In certain instances, the selected cells are asymmetric cells that exhibit particularly desirable characteristics, such as sperm cells, red blood cells, or nerve cells. The particular class of selected cells preferably includes Y-chromosome-bearing sperm cells (which correspond to male sperm) or X-chromosome-bearing sperm cells (which correspond to female sperm).

[0250] In some instances, pulsed beams are used for sorting. In other instances, continuous wave beams are used for sorting. The amount of energy delivered to the focal spot is determined by the effective focal area (cm 2 ) calculated by laser pulse energy (Joules) per J / cm 2 It can be described in terms of peak fluence in units of J / cm. In one example, particle / cell ablation is achieved with a peak fluence of 0.1-100 J / cm. 2 In another example, the selective laser is adapted to cause a phase change of a liquid at the focal point to a gas. In this example, the peak fluence is 10-1000 J / cm 2If the particle / cell is also in the region of focus, it can be about 0.1 J / cm 2 Higher peak fluences can result in particle / cell ablation. Ablation can be considered the process of transferring enough energy to a particle to permanently inactivate it. In the context of biological cells, this can include rendering the cell inviable for its normal function or purpose. For example, sperm cells can be ablated to rapidly induce permanent immobility, or sperm cells can be ablated to "prime" them so that they cannot survive downstream processes such as freezing and thawing. In the former case, ablation can involve rupture of the cell surface membrane, disrupting the integrity of the cell. During "priming," the cell surface membrane remains substantially intact, even though motility may be reduced or halted.

[0251] In some examples, the aperture number of an optical component, such as an objective lens, used to provide the sorting beam ranges from 0.25 to 0.7. The wavelength of the sorting beam may range from 300 to 600 nm, with specific examples being 480 to 580 nm and 300 to 400 nm. In one example, the sorting beam wavelength is 532 nm + / - 5 nm. Alternatively, the sorting beam wavelength may be 355 nm + / - 5 nm.

[0252] The preparation station 105 may also include equipment for staining a batch of cells, e.g., sperm cells collected from a bull. Various other preparation steps, such as diluting the semen sample batch or adding media components known to those skilled in the art, may be performed. In one example, the examination region 125 may include a means for detecting the characteristics of each of a plurality of particles so that the particles can be identified. To this end, fluorescent dyes such as fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC), and peridinin-chlorophyll-protein-based dyes, as well as Alexa Fluor dyes and green fluorescent protein (GFP), which may have excitation and emission wavelengths within the UV spectrum (or other regions of the electromagnetic spectrum, assuming the light source and detectors are appropriately configured), may be deployed to probe individual particles as they pass through the examination region 125. As an example, if the particles are cells, viability assays such as calcein-AM and ethidium homodimer-1 may be used to separate viable from nonviable cells. Alternatively, the cells may be stained with Hoechst 33342, a fluorophore routinely used to stain DNA in mammalian sperm that carry X and Y chromosomes. In these examples, the interrogation region 125 may identify the predominant emission wavelength from a given cell, and the sorting device may act to sort the cells within the microfluidic stream accordingly.

[0253] In one example, the analysis unit 135 may be integrated with the CPU of the computing device, or may be separately integrated with the detector 130 or the sorter 140. In an example, the analysis unit may be implemented as a dedicated logic circuit, such as an FPGA or ASIC. In one example, the sorter may include a radiation source (or nudge laser) and may be configured to optionally direct radiation at each particle to impart at least one of a force and a torque to each particle. Particle movement may involve at least one of displacing and orienting (or nudging) each particle relative to an axis defined by the direction of fluid flow along the microfluidic stream. To this end, the sorter 140 may be configured to direct radiation to a particle when the particle's identification information indicates that the particle should be irradiated, e.g., into separate particle flow streams intended for different collection branches of the microfluidic stream. In an example, particle movement may be achieved by applying an electrostatic charge, as in FACS (fluorescence-activated cell sorting), by buoyancy, by magnetic activation, as in magnetic-activated cell sorting, etc. In instances where the particles are unwanted cells or particles, the radiation directed at the particles may be calibrated to ablate the particles and damage, kill, or reduce the functionality of the particles / cells. In such instances, it may not be necessary to separate the ablated particles from the non-ablated particles.

[0254] The flow controller 110 receives a particle stream 107 from the preparation station, which contains a solution of cells from a prepared batch, e.g., an aqueous solution. The flow controller 110 also receives a sheath stream 108, which may also contain an aqueous solution. In one example, the flow controller 110 combines the particle stream and the sheath stream to create a controlled laminar flow containing the cells, with the sheath stream extending coaxially around the particle stream to form a microfluidic stream. The flow rates of the particles 107 and the sheath stream 108 may be controlled, and the flow controller 110 may include components configured to control the particles and sheath streams to orient and / or confine the cells within the laminar flow. Because some cells are asymmetric, orienting them in a preferred plane improves their interaction with downstream devices, such as the detector 130 and sorter 140. Furthermore, confining the cells within a narrow channel improves the likelihood that the downstream illuminator 120 will strike them as intended.

[0255] The flow rates of the particles 107 and sheath 108 streams may be controlled, and the flow controller 110 may include components configured to control the flow of the particles and sheath to orient and / or confine cells within a laminar or microfluidic stream. In particular examples of the present disclosure, the sample flow rates of the microfluidic streams range from about 0.1 μl / min to about 20,000 μl / min, and from about 10 μl / min to about 5,000 μl / min. In other examples, the flow rate of the microfluidic stream is greater than about 100 μl / min, greater than about 500 μl / min, greater than about 1000 μl / min, greater than about 2000 μl / min, greater than about 4000 μl / min, greater than about 5000 μl / min, greater than about 6000 μl / min, greater than about 7000 μl / min, greater than about 8000 μl / min, greater than about 9000 μl / min, greater than about 10000 μl / min, greater than about 11000 μl / min, greater than about 12000 μl / min, greater than about 15000 μl / min, and / or greater than about 20000 μl / min.

[0256] In some instances, the flow environment through which the microfluidic stream flows is a volume of gas. This can facilitate higher flow rates compared to using microchannels due to reduced friction with the surfaces of the microfluidic channel. Higher flow rates reduce the overall sorting and separation time for each particle, which, in the case of biological cells, reduces their degradation before they can be more optimally preserved, such as by freezing. Faster flow rates also increase the overall performance of the system, allowing larger and / or more samples to be effectively processed. Reducing the surface friction of the microfluidic stream can also improve sorting and / or separation accuracy by reducing undesirable internal hydrodynamic forces that can displace particles in unintended ways.

[0257] The effectiveness of the flow control device 110 to orient and / or confine cells can improve the efficiency of the overall sorting system 100. An example of a flow control device for improving cell orientation and / or confinement is a delivery tube as described in International Patent Publication No. 2020 / 013903, which is incorporated herein by reference. An exemplary flow control device, according to one example, is described with respect to FIG. 3. Other arrangements can alternatively be used; for example, the flow control device may be part of a cytometer.

[0258] Some example methods and devices for flow control devices are useful for achieving orientation of non-spherical particles, such as sperm cells. In one example, the angle of the non-spherical particles can be controlled to achieve a desired angle relative to one or more radiation beams that can be used to inspect and / or sort the particles. In one particular example, the particles are oriented via hydrodynamic and / or radiation pressure-based orientation means. This optimizes the inspection and sorting of particles, such as sperm cells, where radiation absorption and / or emission can be highly orientation-dependent. Therefore, it may be desirable to orient a preferred facet of an asymmetric particle toward the incident radiation beam, the facet having the largest or smallest surface area or some other characteristic.

[0259] In one example, the microfluidic stream 115 emerges from the aperture 113 of the flow control device 110 into a flow environment. The flow environment may be liquid or gas, or a combination thereof. The liquid and / or gas environments through which the microfluidic stream flows may be static or may include movement. The microfluidic stream may emerge in a downward or gravity-driven direction, or at an angle relative to gravity, e.g., vertically or generally upwardly. In one example, the flow environment is bounded by a microfluidic channel, microchannel, or conduit that allows the microfluidic stream to move in a controlled manner from the aperture and confines the flow of particles as they pass through the inspection region or station 125 and the sorting device 140, 143. In some examples, the flow environment may include a liquid of substantially the same viscosity as the liquid in the microfluidic stream. In some examples, the flow environment is a liquid of higher viscosity than the microfluidic stream. In some examples, the fluid in the flow environment may move in the direction of movement of the microfluidic stream. The velocity of this movement may be the same as or different from the velocity of the microfluidic flow. In an alternative example, the flow environment includes a gaseous environment. In the example of a liquid and / or gaseous flow environment, the microfluidic flow does not need to be bounded by a channel or conduit. This approach has many advantages, reducing friction with the conduit, improving laminar flow, and allowing for higher flow rates. The inventors have found that when an aperture engages a channel or conduit, the size of the aperture must be aligned with the internal dimensions of the conduit to ensure that the microfluidic flow maintains laminar flow and has minimal turbulence. The dimensions of the conduit do not always match the dimensions of the aperture, limiting the range of aperture dimensions. Using a flow environment allows for flexibility in the size and shape of the aperture. In particular, using an aperture smaller than a standardized conduit can reduce the proportion of sheath flow compared to particle flow. This reduction in sheath volume thereby increases the concentration of selected cells.The omission of the conduit also reduces refraction of the inspection beam 123 and, if used, the sorting beam 143 through the conduit walls, resulting in improved inspection and sorting accuracy.

[0260] In one example, devices and methods are provided for separating cells within a microfluidic stream in a gaseous environment. Some examples address effects that arise when particle inspection or sorting occurs in a microfluidic stream in a gaseous environment. For example, upon entering the cylindrical profile of a microfluidic stream, light or other electromagnetic radiation (EMR) can be refracted due to the mismatch in refractive index between water and air, which can then distort the beam. The inventors have also found that there is astigmatism from a cylindrical flow profile that provides two focal points (one vertical and one horizontal). This effect causes disruption of the beam entering the stream to inspect or sort particles.

[0261] Figure 10a shows an intensity distribution plot depicting a cross section of a microfluidic stream generated using a modeling program, e.g., Python / NumPy. The X and Y axes represent the width and length of the cross section of the stream. The nominal focus of the beam is at X=Y=0, the center of the circle. It can be observed that there are two foci, with the most intense focus (the "horizontal focus") offset from the center of the stream. The "vertical focus" remains focused around the nominal focus and its surrounding center of the stream. The effect of this phenomenon is that targeting of particles flowing in the Z axis (off-page) is destroyed because the inspection beam 123 and / or the sorting beam 143 are split and defocused. Furthermore, the luminescence propagating from the particles following inspection is destroyed, causing inefficiencies in particle identification and sorting. Figures 10b and 10c show intensity plots as cross sections through the Z and Y axes. Figure 10b shows a close-up of the horizontal focus, and it can be seen that the spot beam is elongated in the Y direction as a result of the refractive index mismatch. Figure 10c shows how the vertical focus of the beam (at the nominal focus) undergoes elongation in the Z axis. This effect occurs because part of the beam intersects the surface of the microfluidic flow from a non-perpendicular direction and is refracted, resulting in a double focus. The actual distance of the center of the horizontal focus from the vertical focus depends on the flow diameter, the refractive index of the fluid, and the aperture number of the objective lens.

[0262] Due to this unusual effect of beam defocusing and vertical elongation as it enters the microfluidic flow, the inventors developed an optical enhancement that allows optimized targeting of the inspection and / or sorting beam to particles flowing in the Z-axis.

[0263] In one example, a method is provided for applying a radiation beam to particles flowing in a microfluidic stream, where the beam crosses a refractive index boundary before entering the microfluidic stream, and the particle flow path is adjusted and / or displaced to be offset from the center of the flow path. This has the effect of moving the particles toward the higher intensity radiation of the horizontal focus of the incident inspection or sorting beam. In another example, the invention includes a method for applying a radiation beam to particles flowing in a microfluidic stream in a gaseous environment, where the horizontal focus is adjusted to be at or near the center of the circular cross-section of the substantially cylindrical microfluidic stream. This has the effect of modifying the focus and increasing the radiation pressure intersecting the particles in the stream. Both examples have the effect of ameliorating or eliminating the effect of refractive index boundaries, thus increasing the intensity of the laser power intersecting the particles and thus achieving improved sorting. In the above examples, the applied radiation can be optimized for nudging, inspection, vaporization, or ablation. The microfluidic stream in the above examples may be in a gaseous environment, or in a solid tube or channel, or any other environment that creates a refractive index boundary that refracts the radiation beam.

[0264] In some examples, the use of an adjusted beam focus allows a user to minimize the amount of power used to generate the laser and applied to the particles. This can have benefits in minimizing heating of optical components and in microfluidic flows, reducing the need to displace particles and / or adjust the microfluidic flow. Minimizing the power directed at the flow can also minimize collateral damage to other flowing particles and allow for control over whether cavitation bubbles form.

[0265] In one example, the inspection and / or sorting beam is tailored to form a beam shape with an unequal aspect ratio, e.g., an elliptical beam shape. This beam shape allows for optimization of the energy applied to particles while spreading the beam across the flow path of the trapped particles in the microfluidic flow. This approach aims to improve beam interaction with particles when desired and reduce missed beam interactions with particles when desired. Tailoring the beam shape in this way may also provide more uniform interaction with a higher percentage of particles compared to a spot beam, which may have high intensity in small areas and very low intensity in other places.

[0266] In some instances, the beam width of the inspection and / or sorting beam is between 1 and 500 microns. In instances where a more confined flow is achieved, the inspection and / or sorting beam width is between 5 and 100 microns.

[0267] In addition to this double-focusing effect caused by the different refractive indices of the flow environment and the microfluidic flow, we also observed that the focus of a beam entering a microfluidic flow is misaligned compared to a beam entering the flow from a fluid of the same refractive index. Figure 11 shows the region of intensity when the beam is focused at the nominal focus X. Figure 12 shows the region of intensity when the nominal focus is adjusted off-center from the center of the flow cross-section. It can be clearly observed that the off-center focus provides a larger region of uniform intensity within the flow.

[0268] When applied to examining or sorting cells in a microfluidic stream, the inventors have found that this lack of beam intensity at the nominal focus means that cells that should interact with the beam may not. This is particularly true when cell confinement is low, i.e., when the spread of cells in the X or Y axis throughout the stream is high, because a particular cell is unlikely to fall outside the region of optimal intensity. Therefore, to achieve the desired interaction between the beam and the majority of particles, higher beam power may be required, which may adversely affect some particles. To address this issue, the inventors have taken the unusual step of intentionally adjusting the focus beyond the nominal focus, e.g., moving the focus "X" as shown in Figures 11 and 12. This adjustment has the effect of increasing the intensity around the center of the stream while also providing a more uniform and widely distributed intensity across the entire cross-sectional area of ​​the stream. Defocusing or diffusing the beam may also, or alternatively, be used to achieve a similar effect.

[0269] In one example, as an alternative or additional feature to the above-described method, a method is provided for applying radiation pressure to particles flowing in a microfluidic stream in a gaseous environment, wherein the focal point of the radiation pressure is adjusted to be off-center of a cross-section of a substantially cylindrical microfluidic stream. The focal point may be at a point within the stream that achieves a maximum region of intensity above a threshold. The focal point may be adjusted to be offset from the center of the cross-section of the stream in a direction defined by the beam propagation and an axis defined by the beam trajectory, the focal point being offset by a distance of approximately 10% to 30% of the stream diameter. In some examples, the offset distance is approximately 15% to 25% of the stream diameter. This technique, referred to herein as "nominal focal offset," has the effect of increasing the radiation pressure intersecting particles in the stream. It also has the effect of increasing the intensity of laser power across the particles, thus resulting in enhanced inspection and sorting.

[0270] In some examples, an inspection metric is measured as a result of the inspection beam interacting with at least one of the microfluidic stream and one or more particles within the stream, and the inspection metric is passed to a control system for processing, and an output signal then adjusts flow parameters based on whether the inspection metric is above or below a particular threshold. In one example, the output signal causes a change in one or more of positioning, flow rate, confinement, or orientation. In one example, the inspection metric is one or more of fluorescent pulse width, intensity, and the ability to distinguish particle characteristics or populations.

[0271] In a further example, the illuminator 120 and / or the sorting device 140 include a diffractive optical element (DOE) that diffuses the inspection beam 123 and / or the sorting beam 143 to achieve a more uniform intensity across the cross-section of the microfluidic stream. In one example, the DOE provides a uniform intensity profile across the distance of the confined particle stream. In one example, the DOE projects a substantially "top hat" profile of each beam 123, 143 within the microfluidic stream.

[0272] In some examples, the focus and / or profile of the inspection and / or sorting beams may be dynamically adjusted. This may depend on an estimated position of the particle stream within the sheath flow of the microfluidic flow. The position of the particle stream may be estimated using area mapping phase shift (AMPS) techniques, and the focus may be set depending on its estimated position. For example, the focus may be set at a predetermined distance beyond the estimated position. This may be achieved using mechanical adjustment of optical components used to provide the inspection beam 123 or sorting beam 142. Alternatively, the position of the particle stream may be adjusted to have a predetermined relationship to the focus. For example, the particle delivery device 110 may be adjusted based on the estimated particle stream position so that its position is adjusted to a desired relationship to the focus.

[0273] In another example, the focus of the inspection and / or sorting beam is adjusted to be offset from the center of the microfluidic stream. In one example, the offset is 15-25% of the stream diameter to account for refraction.

[0274] To solve or at least ameliorate the above-mentioned problems associated with refraction and bi-focusing, the inventors surprisingly discovered that placing the illuminator 120 at a specific distance from the fluid stream allows for accurate inspection and sorting. This "working distance" is measured from the emission side of the final optical component forming the illuminator to the focal point of the beam within the microfluidic stream. When using microfluidic streams in a gaseous environment, it is generally desirable to maximize the working distance to reduce the likelihood of droplets contacting the optics through which the inspection and / or sorting beams propagate or the detector where the emitted light is detected. In one example, a nominal focus offset is achieved by adjusting the position of the lens to account for defocus and achieve uniform beam intensity.

[0275] In one example, the illuminator emits an inspection beam and the sorting device emits a sorting beam, and both beams propagate through a single objective lens (optical component). In another example, the inspection beam and the sorting beam may pass through separate respective optical components as well as at least one shared optical component.

[0276] In one example, the working distance is at least 5-50 mm, and in some examples, the working distance is greater than 10 mm and less than 40 mm. We have calculated that in some examples, the working distance is 10-30% of the flow diameter to achieve an optimal offset from the flow.

[0277] When the flow environment includes a gaseous fluid environment, the gaseous fluid includes a gas, which may include one or more gases or gas mixtures selected from the group consisting of air, nitrogen, carbon dioxide, methane, or one of the noble gases, such as helium, argon, neon, xenon, or krypton. In particular examples, the gaseous fluid is an inert gas, such as nitrogen or a noble gas. Without being bound by theory, it is believed that these inert gases reduce oxidation and contamination of the fluid stream, which may result in increased cell viability after downstream processing.

[0278] The gaseous fluid environment may be maintained at a fluid temperature and / or fluid pressure that improves at least one of cell throughput, detection accuracy, sorting accuracy, or cell health. Cell health in this context refers to the likelihood of survival, motility, or viability for the sorting process. In one example, the temperature is between 18 and 37°C. Maintaining the temperature above 20°C can help ensure minimal viscosity, maintaining flow and reducing blockages. In an alternative example, the temperature is maintained below 15°C. Operating below this temperature can minimize the effects of thermal stress on the cells and ensure that cell motility and viability are maximized after they flow through the system.

[0279] The aperture number of the sorting and / or inspection beams can be optimized to maintain an adequate working distance according to the above constraints while at the same time providing a focal point with adequate power for inspection / sorting. The inventors have found that the aperture number of the objective lens can be about 0.1-0.7, or in some cases 0.2-0.4.

[0280] In one example, the beam width is 30 μm to 200 μm when the microfluidic stream exits the aperture and enters the gaseous environment. In certain instances, it may be preferable to maintain a stream width of 70 μm to 110 μm. The inventors have found that these ranges provide a stream width wide enough to accommodate the particle stream when it is combined with the sheath flow. These widths also allow for precise sorting via nudging particles into different flow paths or ablation of unwanted particles using an ablation sorting laser.

[0281] In some examples, the distance from the center point of the aperture 113 aligned with the end surface of the flow control device to the point where the interrogation beam contacts the microfluidic stream is between 25 and 1000 μm. This distance is referred to as the "divergence distance," shown as arrow ED in FIG. 2. A shorter divergence distance, for example, between 50 μm and 500 μm, may be preferred to maintain flow confinement within the flow environment.

[0282] In some examples described herein, a step is taken to adjust the focus or other beam characteristics to account for the cylindrical shape of the flow. In these cases, the divergence distance should be long enough to allow the flow surface to smooth. Following divergence from the aperture, the flow tapers and stabilizes, as shown in Figure 20b. We have found that attempting an inspection with a divergence distance too close to the aperture results in insufficient cell luminescence signals. This is believed to be due to either too little inspection radiation reaching the cell or too little luminescence radiation reaching the detector. This may be due to refraction of the beam through an unstabilized flow. In some examples, the inspection beam includes a converging beam that forms a conical shape 2001, as shown in Figure 20a. If the divergence distance ED is too small, the cone risks being blocked by the underside of the flow control device 2005, as shown in Figure 20a. Also, in some examples, it is desirable to provide a distance that allows the flow to stabilize its cross-sectional shape and form a circular cross-section. These features help to enable the beam to be precisely focused onto particles within the flow and can also help to minimize any unpredictable refraction effects as the beam enters the flow or as the emission exits the flow.

[0283] The underside of the flow control device 2005 can be shaped to define a region above the aperture in the z-direction through which the inspection and / or sorting beams can be directed. This avoids clipping of a portion of the cone of radiation 2001, thereby improving inspection performance. Similarly, the objective lens can be positioned at least partially within this region to allow the inspection beam to focus closer to the aperture. While the region shown in FIG. 20a is in the form of a triangular recess extending above the line of the aperture, other shapes and configurations may alternatively be used for these purposes. This region above the aperture can be defined by the contour of the flow control device, which can include a recess, chamfer, or notch from the end surface where the aperture is provided.

[0284] However, as described in more detail below, some examples of the present invention take advantage of the proximity of the interrogation beam contact point to the aperture, i.e., minimizing the divergence distance. This can provide benefits in terms of reducing flow divergence and loss of sample flow confinement. Figure 21a shows the results of an experiment in which the divergence distance from the aperture to the focus of the interrogation beam was varied. The discrimination resolution was calculated by calculating the separation between the two fluorescence emission peaks, as shown in Figure 21a. Discrimination resolution refers to the ability to distinguish between two cell populations and can be correlated with the amount of overlap between the two peaks; the greater the overlap, the lower the discrimination resolution, as cells within the overlap cannot be distinguished. In this example, the peaks correspond to X and Y sperm cells stained with a DNA-specific stain. It can be observed that the fluorescence intensity decreases at lower divergence distances, which is understood to be a result of clipping of the beam or emission light on the underside of the flow-focusing device. This results in relatively more overlap between the peaks.

[0285] On the other hand, it can be observed that the discrimination resolution decreases at higher divergence distances, with a divergence distance of 100 μm taken as the baseline (100%). Therefore, in one example, the optimal discrimination resolution can be obtained within a divergence distance range that is neither too small nor too large.

[0286] The X cell selection metric also indicates that a higher divergence distance results in a reduction in the number of X cells selected from the combination of X and Y cells. If it is desired to separate X cells from Y cells, this effect on X cell selection adversely affects the sorting efficiency, throughput, and purity of the sorted cell sample. Therefore, the inventors determined an optimal test zone with a preferred divergence distance. In one example, the divergence distance is greater than 25 μm, 50 μm, or 100 μm. In another example, the divergence distance is less than 400 μm. In one example, the divergence distance is between 25 μm and 400 μm, or between 25 μm and 1000 μm. In another example, considering the decrease in fluorescence intensity, the divergence distance is between 50 μm and 400 μm. In another example, the divergence distance is between 50 μm and 250 μm. The optimal divergence distance may depend somewhat on the flow velocity, but is not significantly affected by it. Thus, in one example, the flow velocity is greater than 5 m / s.

[0287] In other embodiments, the flow velocity is between 5 m / s and 20 m / s. The inspection beam and the sorting beam are separated by a distance referred to as the "inter-beam distance" - IBD in Figure 2. In some examples, the inter-beam distance is between 15 μm and 1000 μm.

[0288] Again, it may be desirable to minimize this distance to prevent flow divergence within the flow environment and to minimize the loss of orientation that asymmetric particles moving within the flow may experience. However, the inter-beam distance may depend on the time it takes to detect the emission, identify the particle type, process the data to determine how to sort that particular particle, and then transmit the signal to the sorting beam to generate a beam with the appropriate characteristics for sorting the particle. Following extensive experimental analysis and modeling, the inventors determined the parameters of these phenomena and found optimal inter-beam distance ranges for a range of flow velocities. Figure 13 and Table 1 below show, by example, the range of inter-beam distances (IBDs) for specific flow velocities. In this example, a minimum IBD of 16 μm corresponds to a 1.6×10 -5 1ms with a propagation delay of s -1This is achieved with a sample flow rate of 20 ms. -1 A flow rate of 320 μm requires a minimum IBD of 320 μm. Thus, in some examples, the IBD is between about 10 μm and about 400 μm. In some examples, the IBD is between about 40 μm and about 300 μm, or between about 50 μm and about 200 μm.

[0289] [Table 1]

[0290] In some examples, the microfluidic stream remains intact and continuous until it passes through the inspection and sorting device beam. The microfluidic stream 115 may remain intact until it intercepts one or more collection vessels 160. In an alternative example, the microfluidic stream can become discontinuous and break up into droplets at some point after the sorting device 140. In electrostatic flow cytometers known in the art, the microfluidic stream is intentionally disrupted by an electric charge imparted by an ultrasonic transducer. For example, Cossariza (2017) (see details in Reference 1 below) describes in Section 1.4 how an electric charge is imparted to the stream, which is then retained after droplet breakoff at a predetermined distance from the nozzle orifice / aperture. This distance from the orifice / aperture to the point where the stream becomes discontinuous is called the "breakoff distance." In conventional flow cytometry, an electrostatically charged plate interacts with the charged droplets to deflect their flow direction according to the cell characteristics identified via the inspection device. However, vibrations imparted by the ultrasonic transducer can interfere with detection via the inspection device. Furthermore, when the inspection device interacts with the fluid stream at the point where droplets begin to form, the uneven and irregular surface of the flow stream results in undesirable and unpredictable refraction of the inspection beam and emitted illumination patterns. If the irregularities are predictable, this problem can be ameliorated by using normalization techniques. However, these require computer processing time, which in turn slows down sorting trigger events, thus limiting cell throughput and efficiency.

[0291] Furthermore, intentional disruption of the flow is undesirable when the sorting device does not require the formation of charged droplets. It has also been hypothesized that imparting an electrical charge to cells can have deleterious effects on the cell surface membrane or cell health. For sperm cells, this is hypothesized to result in a reduction in the fertilization capacity of the sorted sperm cells. To avoid these problems with electrostatic cell sorting, some examples do not include a device for inducing droplet formation, such as an ultrasonic transducer.

[0292] In a particular example, the microfluidic stream comprises a continuous flow up to a take-off distance measured from the aperture. The take-off distance may be configured depending on the sorting method used in the example and may be implemented by appropriate control of microfluidic stream performance metrics, such as flow rate and cross-sectional dimensions. In one example, the present invention provides an electrostatic device downstream of the sorting device. In this example, the electrostatic device is configured to attract or repel droplets, resulting in the droplets being collected in a different container from droplets that are a) unaffected by electrostatic sorting or are attracted in the opposite direction to the desired droplets. This configuration and process achieves at least one of a) attracting droplets with undesired particles retained therein and b) attracting droplets that do not contain particles. Both options result in an increase in the concentration of desired particles per unit volume, either by a) removing undesired particles and surrounding liquid from the collected volume of desired particles, or b) removing liquid from the collected volume of desired particles. An increase in the concentration of desired particles in the collection volume can be beneficial, as described below with reference to the vaporization device 150.

[0293] The take-off distance can be adjusted or maintained by controlling parameters or performance metrics such as the flow rate of the microfluidic stream as well as its cross-sectional dimensions.

[0294] In one example, the flow control device 110 comprises one or more features on its interior or exterior surface to increase the take-off distance, which may include hydrodynamic features adapted to smooth the surface of the microfluidic stream as it exits the aperture.

[0295] In a further example, the sorting device comprises a laser for applying at least one radiation pressure pulse to one or more particles in the fluid stream, the laser adapted to apply sufficient power to cause a change in direction of the particles and / or the formation of droplets containing the particles, the required power being determined experimentally.

[0296] In this example, selected particles are irradiated with sufficient laser power to form droplets. The optimal laser power can be determined experimentally. In some examples, two or more laser pulses interact with the flow (one before the particle in the flow and one after). This causes the fluid stream to split both before and after the particle, initiating droplet formation around the particle. As well as generating droplets, the power imparted to the resulting droplets has the potential to deflect them into different flow paths within the flow environment. This allows for the selection and separation of particles from other particles within the microfluidic flow. Using this approach to promote droplet formation can also increase the concentration of particles within the droplets compared to other methods, such as vibration.

[0297] The sheath 170 may partially or completely surround the microfluidic stream 115 and extend substantially parallel to the stream. This sheath protects the stream from gaseous flows that may interfere with stream positioning, as well as contaminants such as dust and dirt particles that may accumulate on auxiliary equipment. Optical components used to provide the inspection and / or sorting beams may be positioned adjacent to the transparent portion of the sheath or within appropriately configured orifices or portions within the sheath. In one example, the sheath includes an air curtain applied substantially parallel to the stream, in a direction substantially aligned with the stream. When viewed in a cross-section perpendicular to the longitudinal or Z-axis of the stream, the air curtain may be linear (adjacent to the stream), semicircular (partially surrounding the stream), or circular (surrounding the stream). The air curtain may be applied via an annular ring incorporating multiple air jets positioned to emit air that shields the stream. In one additional or alternative example, the sheath includes a solid structure made of a suitable impermeable material. The sheath itself may be separated from the surface of the microfluidic stream by a gas interface, protecting the stream from gas flows in the broader flow environment. Alternatively, the sheath may completely surround the microfluidic stream without a gas interface between the sheath and the sheath inner wall. If a gas interface is present, the sheath may include orifices or slots to allow some mixing between the gas inside and outside the sheath, for example, to moderate the gas flow within the sheath. Additionally, the sheath can protect the stream 115 against contamination from dust and other particulates. Furthermore, the sheath can prevent the diffusion of aerosols from the microfluidic vapor into the surrounding environment. Such aerosols can collect on sensitive optics, reducing the ability for accurate sensing. The sheath may also be adapted to cover the optical components comprising at least one of the illuminator 120, detector 130, and inspection optics (130 and 120), and / or the sorting device 140. The sheath 170 may extend partially or completely to the collection reservoir, and optionally includes a sheath branch to accommodate flow separation.The sheath can take the form of an air curtain, a cylindrical or other shaped tube, a series of flat plates, or any other suitable arrangement for shielding the flow. In another example, the sheath comprises a separate component that shields the optics of at least one of the illuminator 120, detector 130, and / or sorter 140. FIG. 14 shows an example of a sheath comprising a separate sheath component 1410 that covers an objective lens 1420. This sheath component 1410 may be combined into a larger sheath 170, or the larger sheath may be omitted with one or more sheath components 1410 used to shield the beam from various optical components onto the microfluidic flow in a gas or liquid environment.

[0298] In one example, the sheath surrounds the microfluidic flow, which begins at the take-off distance and ends at or near the entrance to the collection vessel. In this example, the sheath does not interfere with the inspection and optionally sorting beam or detector.

[0299] In another example, the sheath surrounds the microfluidic stream, which begins at or near the aperture and ends either at the take-off distance or at a point downstream of the sorting laser. In this example, the sheath may include at least one window or aperture, which allows the interrogation beam, sorting beam, or emission from the particles to pass through the window or aperture.

[0300] In some examples, the distance from the microfluidic flow to the sheath is between 5 and 20 mm.

[0301] The sheath may be positioned to completely or partially surround the fluid flow.

[0302] The inventors have found that when the flow environment is gaseous, the gaseous atmosphere within or around the sheath 170 can be environmentally controlled. This provides advantages, including improved cell viability. The environmental control may also be modified to cause some evaporation of the outer portion (sheath flow) of the microfluidic stream 115. The environmental control may include controlling the temperature and humidity of the gaseous atmosphere, as well as introducing a gas flow 175, such as a warm air flow, into or along the microfluidic stream 115 to further promote evaporation. The illuminator 120, detector 130, and sorter 140 may be positioned in sealing engagement with the sheath 170 to improve control of the atmosphere within the sheath.

[0303] In one example, a microfluidic stream can flow within a sheath, conduit, or channel whose cross section can be circular, square, rectangular, triangular, elliptical, or another desired shape. The sheath, conduit, or channel can be formed from any one or more of a polymer, glass, ceramic, or other solid substrate, or can be a preformed component such as a PTFE tube or glass capillary. The sheath, conduit, or channel can have a depth of about 10 μm to about 2500 μm, a width of about 50 μm to about 2000 μm, and a length of about 10 μm to about 200 mm. For example, the depth of the sheath, conduit, or channel can be about 20 μm to about 10 mm, about 30 μm to about 5000 μm, about 40 μm to about 1000 μm, about 50 μm to about 500 μm, about 60 μm to about 100 μm, and about 70 μm to about 90 μm. For example, the width of the sheath, conduit, or channel may be about 50 μm to about 2000 μm, about 60 μm to about 1500 μm, about 70 μm to about 1000 μm, about 80 μm to about 500 μm, and about 90 μm to about 100 μm. For example, the length of the sheath, conduit, or channel may be about 10 μm to about 10 mm, about 100 μm to about 5000 μm, about 1000 μm to about 2500 μm, and about 1500 μm to about 2000 μm.

[0304] A vaporization device 150 may be positioned upstream or downstream of the sorting apparatus 140 to direct sufficient energy into the microfluidic stream 115 to vaporize a portion of the liquid in the stream. A sorting apparatus may additionally or alternatively include a vaporization device. FIG. 15 illustrates an example of a vaporization device 1550 that emits directed energy to vaporize a portion of the stream. The vaporization device includes a laser of sufficient power to vaporize the portion of the stream. FIG. 15 illustrates a portion of the outer layer 1552 of the stream 1515 that is vaporized 1553, while the inner particle or “sample” stream 1551 is substantially unaffected. The laser can be timed to vaporize the stream with few or no cells present to avoid additional energy affecting the cellular health, motility, or viability of desired cells in the sample or particle stream 1551. This can be important if radiant energy has already been applied to cells, causing them to redirect to a different flow path.

[0305] In another example, the take-off distance of the microfluidic stream may be controlled so that droplets are formed after the sorter 140 and only the droplets containing unwanted cells receive vaporization energy by the vaporization device 150. In this example, the vaporization device receives a signal from the detector 130 and the analysis unit directs the vaporization device to implement a timed laser pulse to coincide with the droplet or region of the microfluidic stream to be vaporized.

[0306] If vaporization or evaporation of some of the liquid in the stream occurs, a suitable extractor can be provided in the region of vaporization to remove the vapor from the gaseous environment. The extractor can be a negative pressure air extractor fan or a solid phase hygroscopic material that absorbs moisture without causing air movement that could affect collection or sorting.

[0307] The vaporization and evaporation examples described above have the effect of increasing the concentration of desired cells in the collected medium in collection vessel 160, which provides several advantages. As described above, this can be achieved by reducing the proportion of sheath flow 1552 compared to particle flow 1551, promoting evaporation along flow 1515, applying vaporization energy 1550, or a combination thereof.

[0308] Concentrated bovine X cells are typically provided in standardized units called "straws," each containing a standard number of X cells. Therefore, increasing the concentration of X cells in the output fluid while maintaining a constant flow rate allows, for example, for the production of more standardized straws per hour. If the concentration of cells in the output fluid is lower than desired, a reconcentration process step may be required to reconcentrate the cells. By reducing the concentration as described herein, this can reduce the need for such downstream processing. One such processing step is centrifugation, which can affect cell health and motility. Processing steps following collection are also time-consuming, exposing sensitive cells to unfavorable temperatures for extended periods. Centrifugation and other concentration processes can cause twisting or other mechanical forces that can damage cells. A "companion" effect has also been noted, where sperm cells in close proximity to other sperm cells have higher viability, motility, and fertility. Thus, some examples may provide a solution to reconcentration problems affecting sperm by increasing the concentration of cells in the output fluid, thereby avoiding or ameliorating the need for reconcentration.

[0309] In some examples, at least one illuminator, such as an IR or UV illuminator 120, may be oriented to deliver illumination substantially perpendicular to each cell passing through the examination region 125. At least one, preferably two or more, detectors 130 may be oriented to absorb light emitted from the particles. In one example, two detectors are positioned perpendicular to each other to capture responsive fluorescence emitted in different directions from the illuminated cells. While the unique architecture of the example using fluid flow in a gaseous environment may allow the detectors to be positioned perpendicular to each other, the planar chip design only allows for detection and examination from a single axis (i.e., at the shortest distance through the planar chip). The detectors may be photomultiplier tubes at any suitable angle relative to each other and the direction of the flow 115, for example, 90 degrees relative to each other and the direction of the flow. The detectors may be photomultiplier tubes or other detectors positioned to collect pulses of responsive fluorescence from the stained particles. In one example, the detectors include avalanche photodiodes. In one example, the detectors are driven by a voltage driver that drives the detectors. For example, a control voltage can be applied to the detector, and an adjustment voltage can be applied to adjust the gain of the analog output. An amplifier can optionally be connected to the detector output to amplify and, optionally, normalize or rectify the output signal. This amplifier is particularly important when detecting luminescence from cells due to the very fast flow rates and low luminescence signals that can be detected from stained and fluorescent cells. The output signal is sent to the analysis unit 135 as described above.

[0310] Alternatively, in the absence of cell staining, IR lasers are used, such as quantum cascade lasers (QCLs), which provide high spectral power densities at specific wavelengths in the mid-infrared and THz ranges corresponding to molecular bond vibrations. In this case, detectors are tuned to detect transmitted or scattered light at different angles.

[0311] One additional optical illumination method that can be applied to the previous configurations disclosed herein adds polarization as a sensing modality to IR-based examination of particles in a flow stream. When molecules examined by mid-infrared vibrational spectroscopy are arranged in a specific manner within the particle being measured (e.g., DNA in a helical configuration), the measured absorption at the molecule's absorption band depends on the polarization of the mid-infrared light. Thus, in one example, an illuminator generates left- and right-circularly polarized light and measures the difference. The observed difference, known as vibrational circular dichroism (VCD), may provide a particularly sensitive measurement of chiral or helical molecules and / or provide information about the folding or configuration of specific particles / molecules within the analyzed particle / cell / droplet. If particles can be successfully distinguished by the detector and analyzer, a sorting device can act to sort particles according to their molecular characteristics. When two detectors are used, this results in two signals or channels of pulsed measurements that are proportional to the intensity or power of the received signal or fluorescence and correspond to a specific angle or angles between the detector directions.

[0312] In one example, the inspection beam and the sorting beam co-propagate through a single or common optical objective, such as an objective lens (or lenses) or a convex mirror, to interact with the particles and the microfluidic stream. The common optical objective is the final optical focusing component closest to the object, in this case the particles in the microfluidic stream. Advantages of this feature of the invention include: Reduction of optical components (e.g., objective lenses) around the aperture, inspection, and sorting regions, which allows for a better view of the flow for alignment and observation purposes and also facilitates positioning the inspection beam closer to the aperture to reduce divergence distances. · Multiple lasers are generally focused to a single focal point, making beam alignment easier. Minimize optical aberrations and enhance focusing ability when focusing through multiple phases, e.g., air, liquid and / or solid window materials.

[0313] Sharing optical components reduces the complexity of the device and simplifies the setup in which the inspection and sorting beams must be directed to focus on the desired portion of the microfluidic stream—for example, the inspection beam is focused to the desired divergence distance, and then the sorting beam is focused to the inter-beam distance. This advantage is enhanced by the short distances involved in the operation of the exemplary device. Beam positioning can be further simplified by angling the beams relative to each other as they propagate toward a common optical objective. The direction in which the two beams intersect the objective determines where their focal points form within the microfluidic stream. As described in more detail below, the inter-beam distance can be easily configured by adjusting the angle between the beams. The divergence distance can be configured by adjusting the angle at which the inspection beam intersects the common objective. Alternatively or additionally, the inspection beams may intersect the common objective at 90 degrees, such that it is the position of the objective that sets the divergence distance. The interbeam distance can be adjusted by adjusting the angle between the co-propagating inspection and selection beams before they intersect with a common objective lens.

[0314] The interbeam distance can be adjusted by modifying the angle of the beams propagating from the final objective. Methods for achieving co-propagation of the inspection and selection beams through the same objective include using dichroic mirrors that pass certain wavelengths but reflect others. Alternatively, if the selection and inspection beams are of similar or identical wavelengths, the beams can be polarized, and a polarizing mirror can be used to reflect one polarity of the beam while passing the other.

[0315] Figures 22a-c show examples of co-propagating beam arrangements of the present invention. In these examples, an inspection beam 2205 (solid line) propagates through an optical objective lens 2210, which focuses (2215) the inspection beam toward a microfluidic vapor 2220 emitted from an aperture 2225 of a flow control device 2230. A co-propagating sorting beam (short-dashed line) 2235 co-propagates through the same (common optical) objective lens and is focused (2215) onto the microfluidic stream 2220 at a downstream location. The divergence distance ED can be observed as the distance between the bottom surface of the flow control device and the interaction point of the inspection beam with the stream. The inter-beam distance IBD can be observed as the distance between the interaction point of the inspection beam with the stream and the interaction point of the sorting beam with the stream. An optional third illumination beam is shown (long-dashed line) 2240, which is broadly focused to illuminate the inspection / sorting region. The illumination beam provides light that allows imaging of the inspection / screening area.

[0316] 22a illustrates a sorting operation in which radiation pressure causes a change in direction of a flow within a gaseous fluid environment or of selected particles within the flow. Cells are collected in a first or second container 2222. Particles are selected according to properties determined following inspection with an inspection beam. Although the particles are shown in a gaseous fluid environment, the sorting operation may also be performed in a liquid fluid environment where the flow is contained by a solid microfluidic chip.

[0317] FIG. 22b shows an arrangement in which the focused beam 2215 passes through a window 2235 and the microfluidic stream is contained within a solid-state microfluidic chip. The semi-focused beam can be adjusted to account for the refractive properties of the window 2235. In instances in which a window is used, the sorting process using this configuration may require the further step of modifying the focus of at least one of the inspection and sorting beams to account for any refraction or aberrations caused by the window 2235. An illumination beam 2240 also propagates through the window to illuminate the inspection and sorting regions. Optical correction means may be required to reduce distortion when propagating the inspection beam through a window.

[0318] For example, the objective lens may include a correction collar to prevent distortion. Additionally, an aperture number less than 0.4 can help manage optical aberrations resulting from the use of a window. In some instances, the window is optically flat to avoid beam distortion. Additionally, the window needs to be made of a material that is resistant to the beam and the damage it may cause.

[0319] 22c shows an example of the invention where a microfluidic stream propagates in a gas-fluid environment, where the inspection and sorting beams are focused to interact with the stream while the illumination beam 2240 illuminates the area.

[0320] As mentioned above, one aspect of the present invention is to achieve a divergence distance (ED) within a specific range, e.g., the ranges described above, along with an optimal interbeam distance (IBD) within a specific range, e.g., the ranges described above. The importance of these two distances can be observed in the configurations shown in Figures 22a-22c. The ED is preferably greater than the minimum ED to ensure that the beam does not clip the edge of the flow-focusing device. Similarly, maintaining a relatively low IBD helps particles in the flow remain trapped and aligned. These properties decrease with distance from the flow-focusing device. Maintaining them increases alignment efficiency and the concentration of desired particles selected after inspection. The IBD is preferably adjustable to enable effective discrimination between particles based, for example, on emission intensity, flow velocity, and fluid viscosity.

[0321] One option for optimizing IBD is to position the inspection beam substantially perpendicular to the sorting beam, while still being directed in the z-axis of the flow. For example, one of the inspection and sorting beams is aligned with the x-axis and the other with the y-axis. In this example, the optical components do not interfere with each other and the beams can be easily aligned independently.

[0322] Alternatively, as described above, a co-propagating configuration can be used. A co-propagating configuration reduces the amount of optical components required, but requires a more complex alignment procedure. An example of a co-propagating beam configuration is shown in FIG. 23, where an inspection beam 2305 is reflected from a first beam splitter 2310 to define a first optical path 2315. The optical path optionally passes through a lens 2320 to shape the beam. The inspection beam is then focused by an objective lens 2210 to interact with the flow 2220. An alternative example may include the use of a window, as shown in FIG. 22b.

[0323] The second sorting beam 2325 propagates toward the second beam splitter 2330 and is reflected to define a second optical path that propagates through the first beam splitter 2310 toward the microfluidic stream 2220. Again, any lens can be used to shape the second beam. It can be observed that the positioning of the second beam at 2325 can be adjusted to achieve the desired IBD. Alternatively, IBD can be obtained by first aligning the beams concentrically to be focused at the same location within the stream. Then, at least one of the beams is angled to generate a non-parallel beam that results in inter-beam separation. Thus, the sorting beam and the inspection beam can be angled relative to each other.

[0324] Optionally, third illumination beam 2340 can reflect from third beam splitter 2345 and propagate through second beam splitter 2330 and first beam splitter 2310, optionally through lens 2320 to objective lens 2210, and then illuminate the inspection and sorting regions as described above. Alternatively, third illumination beam 2340 may simply propagate through the second and first beam splitters without the need for a third beam splitter. It can be observed that this unique configuration of beams, splitters, and lenses works synergistically with the flow focusing device to achieve stable, fast, and accurate cell inspection, sorting, and optionally illumination.

[0325] The beam splitter proposed herein includes a coating that reflects some wavelengths of light while being substantially transparent to other wavelengths. The beam splitter may be a dichroic beam splitter, such as a dichroic mirror, or a harmonic beam splitter. The beam splitter may be a short-pass or long-pass splitter. Thus, the configurations described herein can be positioned to transmit and reflect the higher wavelength of the inspection laser and the selection laser. In some examples, the beam splitter is a polarizing mirror. In one example, the inspection beam includes an infrared (IR) or mid-infrared laser, more preferably a mid-infrared quantum cascade laser (QCL). In this example, the beam splitter includes a beam splitter that reflects IR light, such as a Thorlabs DMSP805. In another example, the inspection beam includes a UV laser in the ultraviolet wavelength range. In one example, the UV laser includes a wavelength of approximately 355 nm. In this case, the first beam splitter reflects UV light, for example 355 nm, or in the UV range such as Thorlabs DMLP550, DMLP567, DMLP605, DMLP638, DMLP650, DMSP550, DMSP567, DMSP605, DMSP638, DMSP650, HBSY12, HBSY22, HBSY11, HBSY21, FELH0550, FELH0600, FESH0550, FESH0600, etc.

[0326] In one example, the wavelengths of the inspection beam and the sorting beam are different. In another example, the wavelengths of the inspection and sorting lasers overlap, for example, a 355 nm inspection laser and a 355 nm sorting laser can be used, with each beam polarized in a different plane from the other. In this example, the beam splitter includes a polarizing mirror, such as a Thorlabs PBS12-355-HP or PBS25-355-HP. In one example, the beam splitter reflects UV light and transmits green light. For example, the beam splitter can reflect at approximately 355 nm and transmit at approximately 532 nm. In another example, the beam splitter reflects green light and transmits UV light.

[0327] In one example, the selected beam includes wavelengths of approximately 355 nm, 405 nm, 515 nm, 532 nm, 800 nm, 1030 nm, or 1064 nm. The second beam splitter 2330 can reflect at approximately 532 nm and transmit at approximately 1064 nm and / or 660 nm, allowing the illumination beam to pass for imaging purposes.

[0328] In one example, the system includes an illumination beam at a different wavelength than the sorting and inspection beams to ensure there is no interference with the detector or sorting. For example, the illumination may be an LED emitting at approximately 565 nm, 590 nm, 595 nm, 617 nm, 625 nm, 660 nm, 680 nm, 700 nm, 730 nm, 780 nm, or 810 nm. The system may also include one or more imaging cameras adjacent to the detector.

[0329] In one example, the illumination beam illuminates the inspection and sorting regions along an axis substantially aligned with the camera optical path. This allows for clear, high-quality imaging of cells / particles as they are inspected and, optionally, sorted. Preferably, the camera is aligned to provide guidance for system alignment and can provide feedback information regarding beam size, shape, power, and intensity distribution. In one example, the illumination beam includes wavelengths (colors) that do not overlap with the inspection and sorting beams to enable filtering during or before signal processing.

[0330] In one example, separation of the inspection beam and the selection beam to achieve IBD is achieved by at least one of the following: The first and / or second beam splitter are angled relative to one another, which results in the angle of the beams being non-parallel after the beam splitter, which achieves beam separation. b. The first and / or second beam splitters are parallel and at least one of the beams is at an angle to the other beam, ie, they are non-parallel. c. The objective lens may be moved and the first beam splitter may be tilted. d. Positioning the beams so that they are offset on the beam splitter, where the first and second beam splitters may be substantially parallel.

[0331] In a further example, at least one of the inspection beam and the sorting beam passes through beam expansion optics before reaching the beam splitter. Beam expansion (or beam diameter control) can be important for optimal compatibility with the optical elements used (e.g., spatially spreading the power to mitigate thermal damage). The beam expansion optics may comprise a pinhole. The pinhole can be placed at the internal focus and used as a spatial filter to increase the resolution of the beam profile, for example, using a Keplerian beam expander. The inventors have found that a higher resolution beam profile leads to a more accurate beam profile in the microfluidic flow, and therefore to more accurate / effective / precise sorting.

[0332] In some examples, it is preferable to include a lens 2320 to shape the beam into an elongated shape for improved inspection and / or sorting. In one example, the lens includes a cylindrical lens to flatten and elongate the inspection beam to provide a wider focus. This modification of the beam provides a wider (and thinner in the z-axis) focus, so that the laser energy is more uniform across the width of the stream and each cell experiences less optical intensity fluctuation due to its positional fluctuation. A further important advantage of the thinner focus is that cells can be better resolved spatially and temporally in the z-axis during inspection and detection. The cylindrical lens elongates the beam in the x-axis, ensuring that a uniform distribution of laser energy (flux) exists across a portion of the microfluidic stream. In one example, the laser exhibits a Gaussian power distribution over its length. In some examples, at least one of the inspection and sorting beams is focused into a line, which may be an elliptical Gaussian at the focal plane. We have found that the 1 / e angle is approximately 1-10 μm high (z-axis) in the focal plane. 2It has been found that the beam width of the inspection or sorting beam is 1 / e 2 The beam width (x-axis, perpendicular to the flow and beam propagation) can be approximately 1 to 5 times the width of the microfluidic flow. Therefore, the beam width can be 150 μm to 750 μm. In one example, the beam width and height of the inspection and sorting beams differ by less than 20%.

[0333] In some examples, both the inspection beam and the selection beam propagate through the same cylindrical lens. This configuration has the advantage of allowing the optical components to be positioned close to each other, making the beam profiles concentric or overlapping. Furthermore, the lens shapes both the inspection beam and the selection beam.

[0334] In one example, the microfluidic channel through which the flow flows after emission from the aperture may be circular, square, rectangular, triangular, elliptical, or another desired cross-sectional shape. The flow control device includes both a delivery microchannel and a focusing and / or confinement chamber. At least one of the flow control device and the microfluidic channel may be formed from any one or more of a polymer, glass, ceramic, or other solid substrate, or may be a preformed component such as a PTFE tubing or glass capillary. At least one of the flow control device and the microfluidic channel may have an internal channel depth of about 10 μm to about 2500 μm, a width of about 50 μm to about 2000 μm, and a length of about 10 μm to about 20 mm. For example, the depth of the microfluidic channel may be about 20 μm to about 10 mm, about 30 μm to about 5000 μm, about 40 μm to about 1000 μm, about 50 μm to about 500 μm, about 60 μm to about 100 μm, and about 70 μm to about 90 μm. For example, the width of the microfluidic channel may be about 50 μm to about 2000 μm, about 60 μm to about 1500 μm, about 70 μm to about 1000 μm, about 80 μm to about 500 μm, and about 90 μm to about 100 μm. For example, the length of the microfluidic channel 402 may be about 10 μm to about 10 mm, about 100 μm to about 5000 μm, about 1000 μm to about 2500 μm, and about 1500 μm to about 2000 μm.

[0335] In some examples, various materials and methods can be used to form any of the above-described components of the present disclosure. In some cases, the various materials selected lend themselves to various methods. For example, the microfluidic focusing device or channel, sheath, window, objective lens, support bracket, or various components of the present disclosure can be formed from solid materials, and the channels can be formed by microfabrication, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithography techniques, etching methods including wet chemical or plasma processes, and the like. In one example, at least a portion of the microfluidic channel or flow control device is formed from silicon by etching features into a silicon chip. Techniques for accurate and efficient fabrication of various fluidic systems and devices of the present disclosure from silicon are known. In another example, various components of the systems and devices of the present disclosure can be formed from polymers, e.g., elastomeric polymers such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), and the like. In another example, the channels of the present disclosure can be formed from polymers, glass, ceramic, or other solid substrates, or can be preformed components such as PTFE tubing or glass capillaries.

[0336] Different components can be made of different materials, for example, at least one of the flow control device and the microfluidic channel can be made of an opaque material such as silicon, and the window 2235 can be made of a transparent or at least partially transparent material such as glass or a transparent polymer for observation and / or control of the inspection and sorting process.

[0337] Optionally, the bottom wall, top wall, or sidewall may be formed from an optically transparent material to allow efficient transmission of electromagnetic radiation. Components can be coated to expose desired chemical functionalities to fluids contacting the interior channel walls. For example, components can be manufactured with interior channel walls coated with another material. Materials used to fabricate various components of the disclosed systems and devices (e.g., materials used to coat the interior walls of fluidic channels) can desirably be selected from among materials that will not adversely affect or be unaffected by fluids flowing through the fluidic system (e.g., materials that are chemically inert in the presence of the fluids used within the device). In one embodiment, various components of the present disclosure are fabricated from polymeric and / or flexible and / or elastomeric materials, which can be conveniently formed from curable fluids to facilitate fabrication by molding (e.g., replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify or that spontaneously solidifies into a solid capable of containing and / or transporting fluids contemplated for use in and with the described microfluidic systems. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymer precursor (i.e., a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermosetting polymers, or mixtures of such polymers heated above their melting points. As another example, a suitable polymeric liquid can include a solution of one or more polymers in a suitable solvent, which forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials that can be solidified, for example, from a melt state or by solvent evaporation, are well known to those skilled in the art. A non-limiting list of examples of such polymers includes polymers from the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Silicone polymers, including PDMS, have several beneficial properties that simplify the fabrication of the microfluidic structures of the present disclosure.For example, such materials are inexpensive, readily available, and can be solidified from a prepolymer liquid via thermal curing. For example, PDMS can typically be cured by exposing the prepolymer liquid to temperatures of, for example, about 65°C to about 75°C for an exposure time of, for example, about 1 hour. Silicone polymers such as PDMS can also be elastomeric and therefore useful for forming very small features with relatively high aspect ratios, as required in certain embodiments of the present disclosure. In this regard, a flexible (e.g., elastomeric) mold or master can be advantageous. In a further example, components of the present invention can be formed from recycled or biodegradable polymers, such as polylactic acid (PLA) and polyhydroxyalkanoates (PHAs).

[0338] Measurement of fluorescence from cells can be affected by several factors, including the orientation and confinement of the cells within the examination region 125, the level of retained staining of the cells in the examination region 125, and biological factors such as whether the cells are dead or abnormal. For sperm cells, measurement of fluorescence also depends on the identity of the sperm sex chromosome (X or Y). The difference in measured signal due to the presence of an X or Y chromosome is only about 3%. This combination of factors makes accurate and efficient classification of sperm cells difficult. Signals from the detector may each correspond to a rapid "hit" applied to each cell to produce a detected signal. A pulse-integrated signal can be derived for each cell by integrating the individual responsive fluorescence emission pulses associated with each cell over a predetermined period of time. These pulse-integrated signals for each channel can be generated in the detector device 130 or the analysis unit 135.

[0339] In one example, a measurement value from a channel corresponding to the fluorescence intensity of a cell represents a measurement data point. In some examples, two or more measurements from two or more channels are combined to represent a measurement data point. This may include, for example, pulse integrals of measurements taken from substantially perpendicular directions. A plot of measurement data points from channel 1 (e.g., 0 degrees) and channel 2 (e.g., 90 degrees) is shown in FIG. 2. In a further example, the detectors may be positioned to detect emissions at other angles relative to each other while still being substantially perpendicular to the flow axis. Each data point represents a fluorescence pulse integral level measured in at least one, and in some examples, two or more perpendicular directions.

[0340] The analysis unit 135 of FIG. 1 analyzes these signals or measurement data points to determine whether the cell should be classified as having a first characteristic A (e.g., part of the X or Y population) (e.g., P1 or P2), and if so, controls the sorting device 140 to sort the cell. In some examples, sorting can be implemented by applying radiation pressure to redirect the cells to direct them for collection or disposal. This can be implemented by applying radiation pressure using a nudge laser to nudge Y cells into different parts of the stream, and then separating the two flow paths using, for example, a microfluidic channel. Improving the proportion of cells correctly classified as having a desired characteristic (e.g., sperm cells with X) improves the efficiency of the sorting device. Other sorting methods may alternatively be used, for example, an ablation laser may apply a directed energy pulse to the cells. This allows all other cells that have the desired characteristic B (e.g., an X chromosome) or cannot be classified to remain undisturbed by the sorting device 140. In an alternative example, Y-type sperm cells can be sorted by moving the cells into a different flow path.

[0341] In some examples, the sorting device 140 includes an ablation laser that applies pulses of laser energy, or a "sorting beam," to the stream 115 as unwanted cells pass through. This sorting method overcomes, or at least ameliorates, some of the drawbacks of more traditional sorting techniques, such as charged droplets and charged deflection plates. These conventional methods typically require a flow-charging wire to intersect the microfluidic stream upstream of the aperture and interrogation beam. The intersection of the wire with the stream can cause disruption and turbulence in the stream, which can reduce confinement and orientation and therefore reduce sorting efficiency. In one example, the present invention does not include a flow-charging wire or a droplet generator. Various characteristics of the ablation laser can be optimized, including pulse rate, wavelength, power, beam shaping, and / or pattern. The energy imparted to cells to cause immobility is described as the ablation threshold, which is the minimum amount of energy per unit area required to cause permanent material modification, damage, or ablation. For example, the ablation threshold may correspond to ablating a cell to rupture the cell surface membrane or otherwise damage the cell sufficiently to rapidly induce permanent immobility.

[0342] Alternatively, cells may be ablated using a lower-energy selective beam that does not induce rapid, permanent immobility but corresponds to cells that become immobile after the freeze-thaw process. This ablation method is referred to as "priming" unwanted cells. It involves subjecting cells to electromagnetic radiation sufficient to transfer energy to selected cells below a predetermined ablation threshold, corresponding to rupture of the cell membrane, and above a predetermined priming threshold, corresponding to the cells being nonviable after the freeze-thaw process. An advantage of this approach is that debris from unwanted cells is not released into the flow, which could adversely affect the remaining desired cells. An additional advantage of approaches that do not rupture cell membranes is that less free DNA from ruptured cells is released into the culture medium, making any downstream genetic analysis of the cells more effective.

[0343] 2 shows a portion of another system 200 for sorting and separating cells, the system including a flow controller 210 that emits a microfluidic stream 215 through an aperture 213 toward a collector 260. The system 200 also includes an interrogation beam generator 220, such as an IR or UV illuminator, one or more detectors 230, and a sorting laser 240.

[0344] System 200 also optionally includes a droplet detector 265 that detects whether microfluidic stream 215 breaks up into droplets 217 above a threshold height, i.e., break-off distance, which may be used as a control input to operate system 200, for example, to increase the flow rate of the particle stream and / or sheath stream, if droplets are detected above the break-off distance.

[0345] System 200 also optionally includes a sheath 270 that extends at least partway between flow controller 210 and collector 260, in this example extending to sorting laser 240. The sheath may completely surround flow 215 up to this point, or it may present one or more baffles with an air gap therebetween.

[0346] The flow controller 210 includes a focusing chamber 211 where the particle stream and sheath stream converge, with the sheath stream moving coaxially around the inner particle stream. This focusing has the effect of orienting asymmetric cells in the particle stream and confining the cells within a narrow range of lateral dimensions. The focusing chamber is fluidly coupled to and tapers toward a microfluidic channel 212 that terminates at an aperture 213 in the flow controller 210. The dimensions of the channel 212 can be experimentally determined to establish a stable laminar flow at the aperture 213. In a specific example, the channel 212 includes a length of at least 10 microns to 10 mm from the outlet of the flow focusing chamber to the aperture outlet, where the aperture outlet is defined as a plane perpendicular to the longitudinal or z-axis of the flow aligned with the end of the flow focusing device. In one example, the channel includes a length of 50 microns to 1 mm, which allows laminar flow to be re-established following hydrodynamic orientation and confinement of particles within the focusing chamber. The channels allow for a steady flow of microfluidic fluid when released into a gaseous environment.

[0347] Aperture 213 may have a cross-section that extends in one axis or dimension (the major axis) more than in the perpendicular axis or dimension (the minor axis). For rectangular apertures, this results in a non-uniform aspect ratio, i.e., when the ratio of the length in one direction to the length in the other direction is greater or less than 1, this may be referred to as an unequal aspect ratio. In particular examples, aspect ratio lengths include at least 10 microns to 1 mm, such as 1:100 to 2:3, or 1:50 to 1:10.

[0348] A microfluidic stream 215 is emitted from an aperture having a cross-sectional shape corresponding to the cross-sectional shape of the aperture. When the stream is emitted into free space and not into a solid conduit such as a microfluidic channel, surface tension acts on the liquid, tending to make the cross-sectional shape symmetrical and circular. This effect can occur over a distance 216, after which the stream assumes a stable circular cross-sectional shape. This changing cross-sectional shape creates internal hydrodynamic forces within the stream, which contract along the major axis and cause further alignment of cells not already aligned along the minor axis. This is visualized in Figure 6 and described in more detail below. Thus, asymmetric dimensions of the aperture 213 along the two cross-sectional axes further improve asymmetric cell orientation. Various shapes, such as rectangular or elliptical, can be used.

[0349] The aperture 213 may additionally or alternatively be adjustable so that the shape, size, orientation, or dimensions of the aperture cross-section can be controlled. In one example, the present invention provides a system with an adjustable aperture, adapted for adjustment of at least one of size, shape, or aspect ratio. This may be achieved, for example, by stretching a flexible material with slits that form the aperture. The aperture may be adjusted to control various characteristics of the microfluidic flow 215, such as the flow rate of one or both of the particle and sheath flows, the relative proportions of the particle and sheath flows, the height at which droplets are formed, particle orientation, or particle confinement.

[0350] The aperture 213 may be adjusted to a cleaning mode in which the aperture is at its maximum size while a high flow rate of sheath flow (with or without particle flow) is applied to remove and wash away any particles or other debris that have become stuck around the aperture or other parts of the flow control device 210.

[0351] The flow controller 210 also includes a pressure sensor 267 associated with the focusing chamber, sheath flow, or particle flow. The pressure measurement can be used to control various characteristics of the flow, such as the flow rate, the concentration of desired cells, and the height of droplet formation.

[0352] 3 and 4 show longitudinal and transverse cross-sectional views, respectively, of an example flow control device 300. The transverse cross-section of FIG. 4 is taken through cut line AA of the longitudinal cross-section of FIG. 3. The flow control device 300 includes a delivery tube 330 that fits within a housing 305. In particular, the delivery tube 330 is securely and precisely received within a cavity 310 of the housing 305, the cavity being defined by the interior surface of the housing. The delivery tube 330 includes a lumen 340 for carrying a particle stream 345, which is a moving liquid, such as an aqueous solution, containing particles, such as sperm cells. The lumen 340 opens to a delivery tube inlet 332 at the input end of the delivery tube 330 and to a delivery tube outlet 333 at the distal end of the delivery tube.

[0353] Delivery tube 330 also includes ridges, fins, or protrusions 335 extending longitudinally along the delivery tube. Ridges 335 engage the inner surface of cavity 310 of housing 305 to secure the delivery tube within the housing. Alternatively, housing 305 includes ridges, fins, or protrusions extending longitudinally along the housing that also engage the outer surface of the delivery tube. Ridges 335 may be sized to ensure a friction fit with the featureless wall of cavity 310, or the wall of the cavity may include a corresponding groove in which the ridge sits. Various other mechanical locking mechanisms may alternatively be used. By extending longitudinally, ridges 335 improve lateral positioning of the distal end of delivery tube 330, such that delivery tube outlet 333 is securely and precisely positioned within housing 305.

[0354] Various alternative engagement structures are possible. While the ridges 335 extend longitudinally, they can also be angled relative to the longitudinal axis to form a helical shape along the outside of the delivery tube. Additionally, while the ridges are shown as continuous, they may be discontinuous, with portions engaging the cavity wall at different longitudinal locations. In a further alternative arrangement, the longitudinally extending ridges may extend from the cavity to engage the delivery tube. In this alternative, the delivery tube 330 may or may not have ridges 335 extending to the cavity wall. In yet another alternative, the outer periphery of the delivery tube 330 may be sized to mate directly with the inner wall of the cavity to ensure a friction fit. The outer surface of the delivery tube and / or the inner surface of the cavity 310 may include recesses to form channels between the housing 305 and the delivery tube 330.

[0355] 3 and 4, one or more sheath flow channels 360 are formed between the delivery tube 330 and the housing 305 for carrying a sheath flow 365, such as an aqueous solution. The sheath flow channels 360 may extend from a sheath flow inlet 362 at the input end of the delivery tube and include channels formed between the ridges 335. The sheath flow channels extend along the outside of the delivery tube 330 to a focusing chamber 370 defined by a volume formed within the housing 305 at the end of the delivery tube 330, into which the particle stream 345 is emitted through the delivery tube outlet 333. The focusing chamber 370 is also fluidly coupled to a delivery microchannel 375 having an aperture 313, from which the combined particle and sheath stream, also referred to herein as microfluidic stream 115, is output for downstream processing.

[0356] The central particle stream 345 is surrounded by one or more flow sheath streams 365 in a coaxial arrangement. The shape and size of the focusing chamber 370, the shape and dimensions of the sheath channel 360, and the lumen 340, along with the flow rates of the particle stream 345 and the sheath stream 365, all contribute to controlling the combined fluid flow from the particle delivery outlet 375. Exemplary use cases include controlling the orientation and confinement of particles within the combined particle and fluid stream 360. In some examples, the particle stream and sheath stream may be concentrically arranged. In other examples, the central axis of the particle stream may be offset compared to the central axis of the sheath stream. This may be useful in embodiments where a downstream inspection and / or sorting beam has a focal offset from the aforementioned microfluidic stream 115, as described with respect to FIGS. 1, 12a, 12b, 13a, and 13b.

[0357] As described above, the longitudinally extending ridges or other engagement structures ensure accurate and stable lateral positioning of the delivery tube outlet 333 within the focusing chamber 370. In more conventional arrangements, the delivery needle is introduced into a tapered volume of sheath fluid, but the distal end of the needle is buffeted and moves laterally by the fluid flow, causing the resulting particle stream to move within or partially mix with the surrounding sheath fluid stream, resulting in a poorly oriented and poorly confined particle-fluid stream. This can make downstream processing difficult, imprecise, and inefficient.

[0358] In some examples, precise longitudinal positioning of delivery tube outlet 333 within focusing chamber 370 may also help optimize the control and stability of particle orientation and / or confinement, or other flow characteristics of microfluidic stream 360 delivered from delivery microchannel or aperture 375. In the examples of Figures 3 and 4, this is achieved by sizing ridge 335 of delivery tube 330 to complement the dimensions of cavity 310 of housing 305 to prevent delivery tube 330 from being inserted into the cavity beyond a predetermined longitudinal position.

[0359] The cavity 310 can be divided into several sections, including a first section 310-S1 having a longitudinal cross-sectional shape, such as a rectangle, that is positioned to engage a corresponding first section of the delivery tube 330-S1 at multiple longitudinal positions. The first section of the cavity can have a substantially uniform cross-section along its length, such as a circular cross-section with a constant diameter. In other examples, the cross-sectional shape can be asymmetric to promote some asymmetry in particle flow within the sheath flow. For example, the cross-sectional shape can be elliptical, hemispherical, triangular, or a combination of a smaller and a larger rectangle. This first section S1 of the cavity 310 is used to receive the ridge 335 of the delivery tube 330. The second section 310-S2 of the cavity is tapered to reduce in size as it extends toward the distal end of the delivery tube. The end of ridge 335, which has a larger dimension, prevents the delivery tube from extending beyond this point, thereby ensuring accurate and stable longitudinal positioning of delivery tube outlet 333 within focusing chamber 370. The end of the ridge may be shaped as shown to complement the interior shape of cavity 310 to further improve this positioning. In other arrangements, grooves in the walls of cavity 310 can be used to receive ridge 335, and the length of the groove can be controlled to control the longitudinal position of delivery tube outlet 333 within focusing chamber 370. In contrast, in more conventional arrangements, the delivery needle may be positioned within a tapered volume of sheath fluid, but if the distal end of the needle is not positioned correctly, the sheath flow may interact turbulently with the particle flow, causing undesired mixing, chaotic misalignment of the particle flow, and poor particle orientation and containment.

[0360] The tapered second portion 310-S2 of the cavity 310 may have a taper angle α relative to the longitudinal axis, and the tapered second portion 330-S2 of the delivery tube 330 may have a taper angle β relative to the longitudinal axis. The taper angle can be adjusted to achieve control of the acceleration of the sheath flow in the sheath flow channel portion. The third portion 310-S3 of the cavity may have a uniform dimension extending along its longitudinal length. Similarly, the third portion 330-S3 of the delivery tube 330 may have a smaller but uniform dimension extending along a similar longitudinal length. The portion of the sheath channel 360 formed between these two portions 310-S3, 330-S3 does not accelerate the sheath flow 365, allowing the sheath flow to be stable and laminar, reducing turbulence.

[0361] The fourth section 330-S4 of the delivery tube includes a distal tip that includes a delivery tube outlet 333. The tip may be shaped to enhance particle orientation and / or confinement, as described in more detail below. This tip region may be complemented by a further tapered fourth section 310-S4 of the cavity 310. A focusing chamber 370 is formed in the fifth section 310-S5 of the cavity 310 when the delivery tube outlet 333 is positioned to discharge the particle stream 345 into the sheath stream 365 that enters the focusing chamber 370. The focusing chamber and other components of the flow control device 300 are configured to induce a combined laminar flow of particles and sheath stream exiting the particle delivery outlet 375, with the particles being predominantly oriented along one axis and predominantly confined to a plane containing that axis.

[0362] Additional portions of the delivery tube and / or sheath flow path 360 may be included, or some depicted portions may be removed from some examples, such as, but not limited to, the third portion 330-S3 from the delivery tube, as needed to impart targeted properties to the particle stream, such as containment. Sheath flow paths of different shapes than those illustrated may alternatively be used.

[0363] In some examples, the shape of the portions may vary along the delivery tube and / or cavity to promote some asymmetry of particle flow within the sheath flow of the final microfluidic flow.

[0364] The sheath flow 365 through the sheath channel 360 may be symmetric or asymmetric. For example, a larger volume in the upper half of the sheath channel 360 displaces the particle flow 345 downward. The sheath flow may also be rotated around the delivery tube, creating vortices that may assist in containing the particle flow. Different cross-sectional volumes along the length of the sheath channel 360 allow for fine control of the sheath flow, including flow acceleration and stabilization. The volume of the sheath channel 360 also controls the rate at which the sheath flow passes through the channel. The ridges 335 and the channels formed between the ridges may also act to stabilize the sheath flow, as the sheath flow may be introduced as a turbulent flow from outside the flow control device.

[0365] FIG. 4 shows a cross section through section line AA of FIG. 3 , allowing the input region of the housing 305 and the delivery tube 330 to be seen. It shows four evenly spaced ridges 335 extending from the delivery tube 330, although any number of ridges could alternatively be used. Various other portions of the delivery tube and housing are indicated with the same reference numerals used for those portions in FIG. 3 . One of the ridges 335L is longer than the others and corresponds to a groove 315 in the outer wall of the housing cavity 310. This arrangement ensures that the delivery tube can only be received within the housing in a single orientation, indicated generally by R. In examples with multiple grooves in the cavity wall 310, each for receiving a single ridge 335, the groove 315 for the longer ridge 335L is deeper so that the longer ridge 335L still fits within only one groove to ensure a predetermined rotational alignment of the delivery tube 330 within the housing 305. In an alternative arrangement using grooves on all ridges, one of these grooves may be wider than the other grooves to accommodate a wider, though not necessarily longer, ridge. In a further alternative, a pin and corresponding hole arrangement may be used to precisely index the delivery tube within the housing. For example, a pin may extend through the housing into a ridge on the delivery tube, or the delivery tube or ridge may include a pin that extends through a hole in the housing. In another arrangement, a magnet in one of the delivery tubes or housing may be used in conjunction with another magnet (or metal feature) in the corresponding housing or delivery tube. A variety of other mechanical rotational alignment features may alternatively or additionally be used.

[0366] The externally visible surfaces of the housing and delivery tube may be marked to assist the user in aligning the delivery tube when inserting it into the housing to ensure rotational alignment.

[0367] In some examples, the lateral position of the sheath flow channel 360 and / or delivery tube outlet 333 may be asymmetric to create a particle flow that is offset within the sheath flow around the microfluidic flow emitted from the aperture 313.

[0368] Some examples may utilize different configurations for the flow controller 300 used to provide the microfluidic flow.

[0369] FIG. 5 shows a cross-sectional view of an aperture 513 from one example of a flow control device 510, from which emerges a combined particle and sheath microfluidic stream. The aperture is rectangular, with a length Dx in one (long) axis X that is longer than a length Dy in the perpendicular (short) axis Y. This provides an aperture with an unequal aspect ratio. Referring also to FIG. 6, the shape of the stream 615 can be seen as it exits the aperture 613; the stream has the same cross-sectional shape as the aperture—in this case, a rectangle, as shown in dashed outline on the right. An asymmetric cell 606, with a flattened circular shape, is also shown within the stream 615. As the stream 615 leaves the aperture 613, surface tension forces act on the stream's surface, causing its cross-section to invert to a circular shape. This change in shape induces a hydrodynamic force 618 within the stream along its longest dimension Dx as this dimension shortens. These forces 618 act to orient the cells 606 along what was their shortest dimension Dy. These surface tension effects can synergize with the effect of the focusing chambers 211, 370 to improve cell alignment, which in turn improves cell examination and therefore sorting.

[0370] 6 illustrates how surface tension acts on a microfluidic stream 615 emerging from an aperture 613 having an asymmetric or non-circular cross-sectional area, causing it to assume a circular cross-sectional shape with uniformly distributed surface tension. This action causes hydrodynamic forces 618 within the microfluidic stream to orient asymmetric particles 606 in a preferred direction for interaction with an inspection and / or sorting beam. The change in cross-sectional shape is indicated at 619, as is the changing orientation of particles 606 with this changing shape.

[0371] FIG. 7 shows a cross-sectional view of another example aperture 713 in an inlet arrangement 710, from which a microfluidic stream with combined particle and sheath flows emerges. The aperture 713 has an elliptical shape, with a length Dx in one axis X longer than a length Dy in a perpendicular axis Y. This provides an aperture with an uneven aspect ratio. As with the example of FIG. 5, the longer extension in one direction (X) compared to the extension in the perpendicular direction (Y) creates surface tension effects that change the cross-sectional shape of the stream from that of the aperture 713 to a circular shape as the stream enters the flow environment below the aperture. This, in turn, generates hydrodynamic forces that help orient cells in the desired axis.

[0372] Although rectangular and elliptical shapes are described, other shapes having a longer dimension in one axis compared to the perpendicular axis may alternatively be used. Examples include parallelograms, trapezoids, and polygons. Single-axis asymmetric shapes such as semicircles or triangles may also be used, or the shape may be completely asymmetric along both perpendicular axes.

[0373] The aperture 713 releases the microfluidic stream into the flow environment and, if used, into a containment chamber for downstream processing. In some examples, the width Dx (X-axis) of at least one of the delivery microchannels 375 leading to the aperture or the aperture itself is between 10 μm and 200 μm. Preferably, the width Dx is between 50 μm and 150 μm. In some examples, the width is equal to the depth Dy (Y-axis).

[0374] In one example shown in FIG. 18 and applicable to any of the flow control devices or methods described herein, the delivery microchannel 1801 immediately adjacent to the aperture includes an expanding longitudinal taper, e.g., at the aperture itself, from a smaller delivery microchannel cross-sectional area upstream to a larger delivery microchannel cross-sectional area downstream, where the cross-sectional area is defined perpendicular to the axis of flow. The expanding longitudinal taper can be limited to a short length of the microchannel adjacent to the aperture, with the remainder of the microchannel having a constant cross-sectional area. For example, the short length can be 1-10% of the total length of the microchannel. In an example, the expanding longitudinal taper can be a trumpet-shaped delivery microchannel to aperture transition. This trumpet-shaped delivery microchannel to aperture transition has been found to generate a more stable microfluidic flow 1802 within the downstream flow environment. The sample flow 1810 is surrounded by the sheath flow 1805, and the use of a trumpet-shaped flared aperture design provides improved downstream containment, inspection, and sorting.

[0375] In some examples, the length (Z-axis) of the delivery microchannel 1801 is at least 10 microns to 10 mm from the outlet of the downstream containment chamber to the aperture 313. In this case, the aperture is defined as a point on a plane perpendicular to the z-axis of the flow aligned with the end of the flow focusing device. In one example, the channel length 375 is in the range of 50 microns to 1 mm. This length may facilitate fully developed laminar flow re-establishing the upstream orientation and subsequent orientation and confinement of particles within the containment chamber.

[0376] In one example shown in FIG. 19 , the width is reduced at a downstream point in the delivery microchannel compared to the width at an upstream point in the microchannel. For example, width W2 is compared to width W1 upstream of entry point 1905 of delivery microchannel 1910. This reduction in width from W1 to W2 shown has the beneficial effect of further increasing particle containment. The width reduction may be tapered along a portion of the length of the delivery microchannel, as shown in FIG. 19 , or along its entirety. In one example, W2 is 50%-95% of W1. In a particular example, the width is reduced from 100-150 μm to 50-99 μm. In one particular example, the width is reduced from about 125 μm to about 50 μm.

[0377] The decreasing longitudinal taper of the microchannel shown in FIG. 19 can be combined with the expanding longitudinal taper shown in FIG.

[0378] The cross-sectional shapes of the delivery microchannel 375, its inlet and outlet or aperture 313 may be the same or different and may include the following: circular, oval, triangular, square, or rectangular with various aspect ratios. In one example, the delivery microchannel 375, its inlet and aperture 313 comprise the same rectangular cross section with an aspect ratio greater than 1:1.

[0379] In a further example, the aperture cross-sectional shape includes a square or rectangular shape. Without wishing to be bound by theory, it is understood that cross-sectional shapes with right-angle corners offer benefits in that they provide a substantially flat surface for the interrogation beam to enter the microfluidic stream and for any emitted light to exit the stream. In one example, the stream is discharged into a rectangular microfluidic channel, in which case the shape of the exit aperture and the channel are substantially aligned. This ensures a smooth transition without turbulence. In another example, the microfluidic stream is discharged into a gaseous free-space fluid flow environment. In this case, the stream shape is immediately induced into a circular cross-section by surface tension reshaping effects. If interrogation occurs sufficiently soon after the stream is discharged, the substantially flat surface after discharge is still presented to the interrogation beam, and the advantages of the aperture shape can still be utilized to improve interrogation and detection of cellular emissions. Thus, in one example, the divergence distance is less than 250 μm. In a further example, the divergence distance is less than 200 μm. The flow velocity also determines the distance beyond which the stream reforms into a substantially circular cross-section. Thus, in one example, the flow velocity is greater than 5 m / s. In another embodiment, the flow velocity is between 5 m / s and 20 m / s. The inventors have found that when flow velocities less than 5 m / s are used in a gaseous fluid environment, the flow collapses and vibrations appear in the flow, which have a detrimental effect on the efficiency of inspection and sorting.

[0380] FIG. 8 shows a cross-sectional view of another example of adjustable apertures 813R, 813F in a flow control device 810, from which a microfluidic flow with combined particle and sheath flows emerges. The apertures may have a circular or slightly elliptical cross-sectional shape in the quiescent state 813R, and an elliptical shape in the forced state 813F, with a larger proportion of the length along one axis X compared to the perpendicular axis Y. This can be achieved using a flexible plastic membrane stretched in the X axis to achieve an aperture shape with a more unequal aspect ratio. In one example, size-tunable membrane nanopores or micropores can be used—see, e.g., Roberts, G.S., Kozak, D., Anderson, W., Broom, M.F., Vogel, R., and Trau, M. (2010). Tunable nano / micropores for particle detection and identification: scanning ion obstruction spectroscopy. Small, 6(23), 2653-2658.

[0381] doi:10.1002 / smll.201001129. In different configurations, the cross-sectional shape can remain substantially the same between the rest and forced states, with the apertures varying only in size or cross-sectional area. For example, a combination of different cross-sectional shapes can be used, such as circular, elliptical, rectangular, triangular, parallelogram, and any other shape.

[0382] Adjustable apertures can be used to accommodate different batches of cells that may use different aqueous solutions or have other properties that can affect factors such as orientation efficiency, the rate at which cells can be examined and / or sorted, and flow characteristics that can affect desired cell concentrations and other characteristics. The size and / or shape of the aperture can be controlled in concert with other controllable and / or measurable properties such as flow rate and take-off distance to achieve desired operating conditions for the sorting and separation device.

[0383] Figure 9 shows a side view of an adjustable aperture arrangement with O-rings 913R, 913F that form a circular or elliptical aperture in the rest state 913R. For perspective views, the focusing chamber and channel, as well as the flow below the aperture 913R, are also shown. A force can be applied upward to the O-ring to deform the aperture 913F into the force state. The forced state aperture 913R can retain the same cross-sectional shape but have a smaller diameter.

[0384] Examples include illuminators, detectors, and sorting devices, each containing multiple optical components. When separation of components from a microfluidic stream is required, e.g., when channels or conduits are used, or when a sheath is used to cover the components, the optical architecture may include one or more optically transparent windows to allow optical radiation to pass through and focus the radiant energy onto the particles. This may achieve at least one of the following: torque and / or pressure on the particles; vaporization of a portion of the microfluidic stream for particle sorting and / or concentration and / or droplet formation; and inspection of the particles, e.g., by promoting fluorescence emission. Possible materials include: Si, Ge, ZnSe, and certain polymers. Si, ZnSe (or similar), and polymers may be compatible with visible, NIR, or SWIR (500-1600 nm) optical inspection or manipulation, if desired. ZnSe or similar materials may be used to enable observation, inspection, and possible manipulation with visible light (directing torque, pressure to redirect, or laser-based cell damage or ablation). Another feature may be an anti-reflection coating applied to both sides of each window, such as an AR coating designed for air outside and water inside. Another feature may be a sloped or wedge-shaped window to further reduce the effects of reflections. Another feature may be a channel or fluid flow width that exceeds the spot size of the laser (e.g., QCL), so that slight shifts in the channel or fluid flow position relative to the beam do not generate spurious signals.

[0385] In a microfluidic stream released into a gaseous environment, the light emitted from particles can be affected by the difference in refractive index between the liquid stream and the gaseous environment. Therefore, for optimal detection of the light emitted, one or more detectors can be positioned to account for this refractive effect on the light emitted. In one example, one or more detectors are positioned to collect maximum light emitted from the particles. In one example, two detectors are used, with an angle between the detectors greater than 90° and less than 120°. In another example, two detectors are used, with an angle between the detectors less than 90° and less than 120°.

[0386] Detector positioning can also be affected by destabilization of the microfluidic flow prior to droplet formation. This phenomenon is illustrated in FIG. 16a, where a stable or laminar portion 1656 of the microfluidic flow forms an unstable region 1657 prior to separation of the fluid into droplets 1658. In the unstable region 1657, the radius of the microfluidic flow fluctuates, resulting in a change in the angle of incidence of an incident beam, such as inspection beam 1620, as shown. This results in a change in focus; indeed, hydrodynamic forces within this region can cause turbulence and alter the path of particles entrained therein. This, in turn, can lead to inaccurate inspection and classification of particles, or, if a sorting beam is involved, inaccurate targeting of undesired particles. Therefore, the positioning of the incident beam may need to be located well above this unstable region 1657 to avoid these effects, as the point at which they begin may change.

[0387] In some examples, the length (longitudinal or Z-axis of the flow) of the delivery microchannel 375 is at least 10 microns to 10 mm from the exit of the focusing chamber 370 to the aperture 313, where the aperture is defined as a point on a plane perpendicular to the Z-axis of the flow aligned with the end of the flow focusing device. In one example, the delivery microchannel comprises 50 microns to 1 mm; this length allows laminar flow to be re-established following particle orientation and confinement in the upstream orientation and confinement chamber. The inventors have found that a minimum length of 10 microns is necessary to ensure that the flowing fluid has an opportunity to stabilize and adopt a laminar flow profile before being released from the aperture.

[0388] In another example, shown in Figure 16b, droplet formation can be promoted before the onset of an unstable region by applying a droplet forming or vaporizing beam 1622 to a stable portion 1656 of the microfluidic stream. Greater control over droplet formation can allow for more reliable positioning of the detector and beam forming device and can also reduce the distance over which the system operates. For example, if a vaporizing sorting beam is used, this can be implemented closer to the inspection beam by promoting earlier droplet onset using the droplet forming beam 1622.

[0389] In another example shown in FIG. 17, a detection apparatus 1700 includes one or more illuminators 1720 positioned to direct a beam toward the microfluidic stream 1715 and a detector 1730 positioned to detect responsive emissions from particles 1706 impinged by the beam. One or more interrogation beams may be used with multiple detectors arranged in an arc to collect responsive emissions from particles in the microfluidic stream. In another example, multiple detectors may be arranged linearly, as shown in FIG. 17B. In one example, the beam 1722 may be an infrared or UV beam, and the divergence 1727 may be fluorescent light. The beam 1722 may be controlled to provide an elliptical intensity pattern across the microfluidic stream 1715, although other intensity patterns, such as circular, may also be generated.

[0390] Ideally, particles 1706 may be intended to be well confined within a narrow cross-sectional portion of the microfluidic stream; however, in reality, particles may spread across a larger cross-sectional portion of the microfluidic stream, as illustrated by particle 1706 at three representative locations. Particles at different lateral positions within the stream may result in different divergences 1727 toward detector 1730. For example, fluorescent light 1727 may be transmitted in different directions and / or propagate different distances through the microfluidic stream. The different directions may also result in different diffraction angles through the interface between microfluidic stream 1715 and the flow environment between it and the detector.

[0391] In one example, detector 1730 may comprise multiple detection units 1732, such as photodiodes in a photoarray, positioned over a range of angles from the point where interrogation beam 1722 intersects particle 1706. This range of angles may extend in one or more planes. For example, the photoarray may be positioned to capture emanations from particles at different positions 1706 within interrogation beam 1722.

[0392] The output 1733 from each detection unit 1732 is sent to an analyzer 1735, which is configured to combine the outputs from the detection units into a signal that can be used to classify particles. Different weighting can be applied to some outputs; for example, outputs at the edge of the detector 1730 can correspond to more degraded emissions than others, in which case they can be amplified to normalize the amplitude of the output 1733. Due to different propagation paths, there may be different delays and phases between the different emissions 1727, and the analyzer 1735 can be configured to compensate for these or otherwise normalize the signal. In one example, the circular cross-section of the microfluidic flow causes refraction of the fluorescent emissions during the transition from the medium to a first refractive index to a second refractive index. In this example, independent detection of emission signals at different locations on the array (which may be linear, arc-shaped, or other shapes) allows the analyzer to independently scale at least one of the independently detected emissions to obtain a normalized signal that accounts for the effects of refraction. This results in enhanced signal processing capabilities and greater accuracy in determining cell characteristics and downstream sorting. The detector may be used in conjunction with a focusing objective.

[0393] In some examples, differences in propagation paths and / or refractive properties may be used by analyzer 1735 to improve classification of particles despite different possible refractive properties of particle flow or position within beam 1722. The correspondence between output 1733 and particle classification may be determined, for example, experimentally or using machine learning.

[0394] Any and all references to publications or other documents, including but not limited to patents, patent applications, articles, web pages, books, etc., presented anywhere in this application are incorporated herein by reference in their entirety.

[0395] As noted elsewhere, the disclosed examples are set forth for illustrative purposes only and are not limiting. Other examples are possible and covered by the present disclosure, and will be apparent from the teachings contained herein. Thus, the breadth and scope of the present disclosure should not be limited by any of the above examples, but should be defined only according to the claims supported by this disclosure and their equivalents. Furthermore, examples of the present disclosure may include methods, systems, and apparatus / devices that may further include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding to binding event determination systems, devices, and methods. In other words, elements from one or another disclosed example may be interchangeable with elements from other disclosed examples. Additionally, one or more features / elements of a disclosed example may be removed and still yield patentable subject matter (thus yielding even more examples of the present disclosure). Additionally, some examples correspond to systems, devices, and methods that specifically lack one and / or another element, structure, and / or step (where applicable) as compared to the teachings of the prior art, and thus represent patentable subject matter and are distinguishable therefrom (i.e., claims directed to such examples may include one or more negative limitations to note the absence of one or more features of the teachings of the prior art).

[0396] For convenience and brevity of explanation, different features are described separately, but it will be understood that these features may be combined in different ways in different examples, for example, features of flow control devices such as those shown in Figures 18, 19, 20a, 3, 4, 5, 7, 8, and 9. Similarly, different aspects of downstream processing may be combined, such as those shown in Figures 1, 2, 11a-12b, 14, 17a, 17b, 20a, 20b, 22a-22c, 23, and 24a-24c.

[0397] The various inventive concepts disclosed herein may be embodied as one or more methods (as referred to as such). The actions performed as part of a method may be ordered in any suitable manner. Thus, while the illustrative examples are shown as sequential acts, examples may be constructed in which the actions are performed in an order different from that depicted, which may include performing some acts simultaneously.

[0398] References 1. Cossarizza A, Chang HD, Radbruch A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 2017, 47(10):1584-1797. doi:10.1002 / eji201646632

Claims

1. 1. A method for treating particles in a particle stream, the method comprising: delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic vapor and monitoring responsive emissions from the illuminated particles; thereafter, directing sorting electromagnetic radiation at at least a portion of the particles in the microfluidic stream to sort the particles into at least two populations dependent on the monitored responsive emissions of the particles; The method, wherein the microfluidic flow comprises a continuous phase flow of a liquid.

2. 10. The method of claim 1, wherein the flow environment comprises one or more of the following: a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, and a gaseous fluid environment.

3. The method of claim 2 , wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

4. The selecting step comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and directing the subsequent screening electromagnetic radiation at particles that are biological cells, the subsequent screening electromagnetic radiation being configured to deliver energy to the selected cells that is below a predetermined ablation threshold corresponding to rupturing the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after a freezing and thawing process.

5. The method of any one of claims 1 to 4, wherein the inspection electromagnetic radiation and the selection electromagnetic radiation are directed through a common optical objective.

6. The method of claim 5 , wherein the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through the common optical objective lens at an angle relative to each other.

7. 7. The method of claim 6, wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjusted to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

8. 8. The method of any one of claims 5 to 7, wherein the microfluidic stream is delivered from a flow control device having the microfluidic aperture, the flow control device being shaped to define a region above the microfluidic aperture into which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

9. The method of claim 8 , wherein the objective optical component is positioned at least partially within the region.

10. 10. The method of any one of claims 1 to 9, wherein the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

11. The method of claim 10, wherein the divergence distance is less than 400 μm and greater than one of 25 μm, 50 μm, and 100 μm.

12. 12. The method of any one of claims 1 to 11, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, comprising one or more of the following: 10 μm to 400 μm.

13. 13. The method of any one of claims 1 to 12, wherein at least one of the inspection electromagnetic radiation and the selection electromagnetic radiation is controlled to propagate as a beam that is equal to or wider than the microfluidic stream when it intersects with the microfluidic stream.

14. 14. The method of any one of claims 1 to 13, wherein monitoring responsive luminescence from the illuminated particles comprises using outputs from a plurality of sensors arranged around the microfluidic stream, the sensors being positioned to capture responsive luminescence from different directions.

15. 15. The method of claim 14, wherein the outputs from the sensors are normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

16. The method of any one of claims 1 to 15, comprising adjusting a cross section of the microfluidic aperture.

17. The method according to any one of claims 1 to 16, wherein the flow velocity of the microfluidic stream is between 5 and 20 m / s.

18. 1. A method for treating particles in a particle stream, the method comprising: delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic vapor through a common optical objective and monitoring responsive emissions from the illuminated particles; and then directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream through a common optical objective to sort the particles into at least two populations dependent on the monitored responsive optical emissions of the particles.

19. 20. The method of claim 18, wherein the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through the common optical objective at an angle relative to one another.

20. 20. The method of claim 19, wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjusted to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

21. 21. The method of any one of claims 18 to 20, wherein the microfluidic stream is delivered from a flow control device having the microfluidic aperture, the flow control device beginning to be shaped to define a region above the microfluidic aperture toward which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

22. The method of claim 21 , wherein the objective optical component is positioned at least partially within the region.

23. 23. The method of any one of claims 18 to 22, wherein the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

24. 24. The method of claim 23, wherein the divergence distance is less than 400 μm and greater than one of 25 μm, 50 μm, and 100 μm.

25. 25. The method of any one of claims 18 to 24, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, comprising one or more of the following: 10 μm to 400 μm.

26. The method of any one of claims 18 to 25, wherein the sorting electromagnetic radiation is controlled to propagate as a sorting beam that is wider than the microfluidic stream when it intersects with the microfluidic stream.

27. 27. The method of any one of claims 18 to 26, wherein monitoring responsive emissions from the illuminated particles comprises using outputs from a plurality of sensors arranged around the microfluidic stream using a photoarray with detectors positioned to capture responsive emissions from different directions.

28. 28. The method of claim 27, wherein the outputs from the sensors are normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

29. The method of any one of claims 18 to 28, comprising adjusting a cross section of the microfluidic aperture.

30. 30. The method according to any one of claims 18 to 29, wherein the flow velocity of the microfluidic stream is between 5 and 20 m / s.

31. 31. The method of any one of claims 18 to 30, wherein the flow environment comprises one or more of a microchannel, optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

32. 32. The method of claim 31 , wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

33. The selecting step comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and directing the subsequent screening electromagnetic radiation at particles that are biological cells, the subsequent screening electromagnetic radiation being configured to deliver energy to the selected cells that is below a predetermined ablation threshold corresponding to rupturing the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after a freezing and thawing process.

34. 1. A method for treating particles in a particle stream, the method comprising: delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; directing an interrogation beam at the particles in the microfluidic vapor and monitoring responsive emissions from the illuminated particles; thereafter sorting the particles into at least two populations depending on the monitored responsive luminescence of the particles; The method, wherein a divergence distance between the aperture and the inspection beam is less than 1000 μm.

35. 35. The method of claim 34, wherein the divergence distance is less than 400 μm.

36. 36. The method of claim 34 or 35, wherein the divergence distance is greater than one of 25 μm, 50 μm, or 100 μm.

37. 37. The method of any one of claims 34 to 36, wherein the flow velocity of the microfluidic stream is between 5 and 20 m / s.

38. 38. The method of any one of claims 34 to 37, wherein sorting the particles comprises directing sorting electromagnetic radiation at at least a portion of the particles in the microfluidic stream to sort the particles into the at least two populations.

39. The selecting step comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and directing the subsequent screening electromagnetic radiation at particles that are biological cells, the subsequent screening electromagnetic radiation being configured to deliver energy to selected cells that is below a predetermined ablation threshold corresponding to rupturing the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after a freezing and thawing process.

40. 40. The method of claim 38 or 39, wherein the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through a common optical objective.

41. 41. The method of claim 40, wherein the inspection electromagnetic radiation and the screening electromagnetic radiation are directed through the common optical objective at an angle relative to one another.

42. 42. The method of claim 41 , wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjusted to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

43. 43. The method of any one of claims 38 to 42, wherein the microfluidic stream is delivered from a flow control device having the microfluidic aperture, the flow control device beginning to be shaped to define a region above the microfluidic aperture through which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

44. 44. The method of claim 43, wherein the objective optical component is positioned at least partially within the region.

45. 45. The method of any one of claims 38 to 44, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, comprising one or more of the following: 10 μm to 400 μm.

46. 45. The method of any one of claims 38 to 44, wherein the inspection electromagnetic radiation and / or the screening electromagnetic radiation is controlled to propagate as a screening beam that is wider than the microfluidic stream when it intersects with the microfluidic stream.

47. 47. The method of any one of claims 34 to 46, wherein the microfluidic flow comprises a continuous phase flow of a liquid.

48. 48. The method of any one of claims 34 to 47, wherein the flow environment comprises one or more of a microchannel, optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

49. 49. The method of claim 48, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

50. 50. The method of any one of claims 34 to 49, wherein monitoring responsive emissions from the illuminated particles comprises using outputs from a plurality of sensors arranged around the microfluidic stream using a photoarray with detectors positioned to capture responsive emissions from different directions.

51. 51. The method of claim 50, wherein the outputs from the sensors are normalized to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

52. 52. The method of any one of claims 34 to 51, comprising adjusting a cross section of the microfluidic aperture.

53. 1. An apparatus for treating particles in a particle stream, comprising: a means for delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; means for directing interrogation electromagnetic radiation at the particles in the microfluidic stream and monitoring responsive optical emissions from the illuminated particles; and means for directing sorting electromagnetic radiation at at least a portion of the particles in the microfluidic stream to sort the particles into at least two populations dependent on the monitored responsive optical emissions of the particles; The apparatus, wherein the means for delivering the microfluidic stream is configured to maintain the microfluidic stream as a liquid continuous phase flow when receiving the electromagnetic radiation.

54. 54. The apparatus of claim 53, wherein the flow environment comprises one or more of the following: a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, and a gaseous fluid environment.

55. 55. The apparatus of claim 54, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

56. The means for directing the selected electromagnetic radiation comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; and / or directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and / or 56. The apparatus of any one of claims 53 to 55, configured to direct the subsequent screening electromagnetic radiation at particles that are biological cells, the subsequent screening electromagnetic radiation being configured to deliver energy to selected cells that is below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after a freezing and thawing process.

57. 57. Apparatus according to any one of claims 53 to 56, comprising a common optical objective lens through which the inspection electromagnetic radiation and the selection electromagnetic radiation are directed.

58. 58. The apparatus of claim 57, configured to direct the inspection electromagnetic radiation and the screening electromagnetic radiation through the common optical objective lens at an angle relative to one another.

59. 59. The apparatus of claim 58, wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjustable to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

60. 60. The apparatus of any one of claims 57 to 59, wherein the means for delivering a microfluidic stream comprises a flow controller having the microfluidic aperture through which the microfluidic stream is delivered, the flow controller being shaped to define a region above the microfluidic aperture into which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

61. 61. The apparatus of claim 60, wherein the objective optical component is positioned at least partially within the region.

62. 62. The apparatus of any one of claims 53 to 61, wherein the inspection electromagnetic radiation is configured to propagate as an inspection beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

63. 63. The apparatus of claim 62, wherein the divergence distance is less than 400 μm and greater than one of 25 μm, 50 μm, and 100 μm.

64. 64. The apparatus of any one of claims 53 to 63, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation are configured to propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, comprising one or more of the following: 10 μm to 400 μm.

65. 65. The apparatus of any one of claims 53 to 64, wherein at least one of the inspection electromagnetic radiation and the selection electromagnetic radiation is controlled to propagate as a beam that is equal to or wider than the microfluidic stream when it intersects with the microfluidic stream.

66. 66. The apparatus of any one of claims 53 to 65, comprising a plurality of sensors arranged around the microfluidic stream and positioned to capture responsive emissions from different directions.

67. 67. The device of claim 66, configured to normalize the output from the sensors to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

68. 68. A device according to any one of claims 53 to 67, wherein the cross section of the microfluidic aperture is adjustable.

69. 69. The apparatus of any one of claims 53 to 68, configured to provide a flow velocity of the microfluidic flow of 5 to 20 m / s.

70. 1. An apparatus for treating particles in a particle stream, comprising: a means for delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; a common objective lens; means for directing interrogation electromagnetic radiation through the common optical objective lens to the particles in the microfluidic vapor and monitoring responsive emissions from the illuminated particles; thereafter, means for directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream through the common optical objective lens to sort the particles into at least two populations dependent on the monitored responsive optical emissions of the particles.

71. 71. The apparatus of claim 70, configured to direct the inspection electromagnetic radiation and the screening electromagnetic radiation through the common optical objective lens at an angle relative to one another.

72. 72. The apparatus of claim 71, wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjustable to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

73. 73. The apparatus of any one of claims 70 to 72, wherein the means for delivering the microfluidic flow is a flow control device having the microfluidic aperture, the flow control device beginning to be shaped to define an area above the microfluidic aperture through which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

74. 74. The apparatus of claim 73, wherein the objective optical component is positioned at least partially within the region.

75. 75. The apparatus of any one of claims 70 to 74, wherein the inspection electromagnetic radiation is configured to propagate as an inspection beam that intersects the microfluidic stream at a divergence distance from the microfluidic aperture, the divergence distance being between 25 and 1000 μm.

76. 76. The apparatus of claim 75, wherein the divergence distance is less than 400 μm and greater than one of 25 μm, 50 μm, and 100 μm.

77. 77. The apparatus of any one of claims 70 to 76, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation are configured to propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, including one or more of the following: 10 μm to 400 μm.

78. 78. An apparatus according to any one of claims 70 to 77, wherein the sorting electromagnetic radiation is controlled to propagate as a sorting beam which is wider than the microfluidic stream when it intersects with the microfluidic stream.

79. 79. An apparatus according to any one of claims 70 to 78, comprising a photoarray having detectors arranged to capture responsive emissions from different directions.

80. 80. The apparatus of claim 79, wherein the apparatus is configured to normalize the output from the sensors to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

81. 81. A device according to any one of claims 70 to 80, wherein the cross section of the microfluidic aperture is adjustable.

82. 82. The apparatus of any one of claims 70 to 81, configured to provide a flow velocity of the microfluidic flow of 5 to 20 m / s.

83. 83. The method of any one of claims 70-82, wherein the flow environment comprises one or more of a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, a gaseous fluid environment.

84. 84. The apparatus of claim 83, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

85. The means for directing the interrogation electromagnetic radiation comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; and / or directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and / or 85. The apparatus of any one of claims 70 to 84, configured to: direct the selected subsequent electromagnetic radiation at particles that are biological cells, the subsequent selected electromagnetic radiation being configured to deliver energy to selected cells that is below a predetermined ablation threshold corresponding to rupturing the cell membrane and above a predetermined priming threshold corresponding to the cells being non-viable after a freezing and thawing process.

86. 1. An apparatus for treating particles in a particle stream, said apparatus comprising: a means for delivering a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream comprising a plurality of particles; means for directing an interrogation beam at the particles in the microfluidic vapor and monitoring responsive emissions from the illuminated particles; means for sorting the particles into at least two populations dependent on the monitored responsive luminescence of the particles; The apparatus is configured such that a divergence distance between the aperture and the inspection beam is less than 1000 μm.

87. 87. The apparatus of claim 86, wherein the divergence distance is configured to be less than 400 μm.

88. 88. The apparatus of claim 86 or 87, wherein the divergence distance is configured to be greater than one of 25 μm, 50 μm, or 100 μm.

89. 89. The apparatus of any one of claims 86 to 88, configured so that the flow velocity of the microfluidic flow is between 5 and 20 m / s.

90. 90. The apparatus of any one of claims 86 to 89, wherein the means for sorting the particles comprises means for directing sorting electromagnetic radiation at at least some of the particles in the microfluidic stream to sort the particles into the at least two populations.

91. The means for directing selected electromagnetic radiation comprises: directing the subsequent sorting electromagnetic radiation at particles in one of the populations in the microfluidic stream to impart a radiation pressure to the particles; and / or directing the subsequent electromagnetic radiation at particles in one of the populations in the microfluidic stream to ablate the particles; and / or 91. The apparatus of claim 90, configured to direct the subsequent screening electromagnetic radiation at particles that are biological cells, wherein the subsequent screening electromagnetic radiation delivers energy to selected cells that is below a predetermined ablation threshold corresponding to rupture of the cell membrane and above a predetermined priming threshold corresponding to the cell being non-viable after a freezing and thawing process.

92. 92. Apparatus according to claim 90 or 91, configured to direct the inspection electromagnetic radiation and the screening electromagnetic radiation through a common optical objective.

93. 93. The method of claim 92, configured to direct the inspection electromagnetic radiation and the screening electromagnetic radiation through the common optical objective lens at an angle relative to one another.

94. 94. The apparatus of claim 93, wherein the angle between the inspection electromagnetic radiation and the selection electromagnetic radiation directed through the common optical objective lens is adjustable to define an inter-beam distance between a focal point of the inspection electromagnetic radiation within the microfluidic vapor and a focal point of the selection electromagnetic radiation within the microfluidic vapor.

95. 95. The apparatus of any one of claims 86 to 94, wherein the means for delivering the microfluidic flow is a flow control device having the microfluidic aperture, the flow control device beginning to be shaped to define an area above the microfluidic aperture through which the inspection electromagnetic radiation and / or the screening electromagnetic radiation is directed.

96. 96. The apparatus of claim 95, wherein the objective optical component is positioned at least partially within the region.

97. 97. The apparatus of any one of claims 90 to 96, wherein the inspection electromagnetic radiation and the subsequent screening electromagnetic radiation are configured to propagate as respective inspection and screening beams that intersect the microfluidic stream separated by an inter-beam distance of at least 10 μm, comprising one or more of the following: 10 μm to 400 μm.

98. 98. An apparatus according to any one of claims 90 to 97, wherein the sorting electromagnetic radiation is controlled to propagate as a sorting beam which is wider than the microfluidic stream when it intersects with the microfluidic stream.

99. 99. An apparatus according to any one of claims 86 to 98, wherein said means for delivering a microfluidic stream is configured to maintain said microfluidic stream as a continuous phase flow of liquid.

100. 100. The apparatus of any one of claims 86-99, wherein the flow environment comprises one or more of the following: a microchannel optionally comprising a substantially transparent material, a substrate exposed to a fluid environment, a liquid fluid environment, and a gaseous fluid environment.

101. 101. The apparatus of claim 100, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gas sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

102. 102. An apparatus according to any one of claims 86 to 101, comprising a photoarray having detectors arranged to capture responsive emissions from different directions.

103. 103. The apparatus of claim 102, configured to normalize the output from the sensors to compensate for differences in one or more of the following characteristics of the responsive luminescence captured from each sensor: amplitude, phase, propagation delay, and refraction effects when traversing from the microfluidic flow to the flow environment.

104. An apparatus according to any one of claims 86 to 103, wherein the cross section of the microfluidic aperture is adjustable.

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